Báo cáo khoa học: Complex II from phototrophic purple bacterium Rhodoferax fermentans displays rhodoquinol-fumarate reductase activity pot

Complex II from phototrophic purple bacterium
Rhodoferax
fermentans
displays rhodoquinol-fumarate reductase activity
Hiroko Miyadera
1,
*, Akira Hiraishi
2
, Hideto Miyoshi
3
, Kimitoshi Sakamoto
3
, Reiko Mineki
4
,
Kimie Murayama
4
, Kenji V. P. Nagashima
5
, Katsumi Matsuura
5
, Somei Kojima
6
and Kiyoshi Kita
1
1
Department of Biomedical Chemistry, Graduate School of Medicine, The University of Tokyo, Japan;
2
Department of Ecological
Engineering, Toyohashi University of Technology, Japan;
3

Division of Applied Life Sciences, Graduate School of Agriculture,
Kyoto University, Japan;
4
Division of Biochemical Analysis, Central Laboratory of Medical Science, Juntendo University
School of Medicine, Tokyo, Japan;
5
Department of Biology, Tokyo Metropolitan University, Japan;
6
Department of Parasitology, Institute of Medical Science, University of Tokyo, Japan
It has long been accepted that bacterial quinol-fumarate
reductase (QFR) generally uses a low-redox-potential
naphthoquinone, menaquinone (MK), as the electron
donor, whereas mitochondrial QFR from facultative and
anaerobic eukaryotes uses a low-redox-potential benzoqui-
none, rhodoquinone (RQ), as the substrate. In the present
study, we purified novel complex II from the RQ-containing
phototrophic purple bacterium, Rhodoferax fermentans that
exhibited high rhodoquinol-fumarate reductase activity in
addition to succinate-ubiquinone reductase activity. SDS/
PAGE indicated that the purified R. fermentans complex II
comprises four subunits of 64.0, 28.6, 18.7 and 17.5 kDa
and contains 1.3 nmol heme per mg protein. Phylogenetic
analysis and comparison of the deduced amino acid
sequences of R. fermentans complex II with pro/eukaryotic
complex II indicate that the structure and the evolutional
origins of R. fermentans complex II are closer to bacterial
SQR than to mitochondrial rhodoquinol-fumarate reduc-
tase. The results strongly indicate that R. fermentans
complex II and mitochondrial QFR might have evolved
independently, although they both utilize RQ for fumarate

reduction.
Keywords: rhodoquinone; complex II; quinol-fumarate
reductase; succinate-ubiquinone reductase; Rhodoferax
fermentans.
Fumarate respiration is a common anaerobic pathway
found in both anaerobic bacteria [1] and mitochondria
from facultative anaerobic eukaryotes [2,3]. Quinol-fuma-
rate reductase (QFR) is an integral membrane protein
located in the bacterial cytoplasmic membrane and the inner
mitochondrial membrane. QFR functions as the terminal
oxidase in anaerobic respiration, such as in the NADH-
fumarate reductase system [4], and catalyzes the quinol-
mediated reduction of fumarate to succinate. QFR is also
referred to as complex II, an enzyme complex that catalyzes
the reduction of fumarate as well as the oxidation of
succinate (succinate-ubiquinone reductase, SQR), that is the
reverse reaction of fumarate reduction. SQR is a dehydro-
genase involved in both the respiratory system and the
tricarboxylic acid cycle.
The subunit structure of complex II is conserved among
species, and is composed generally of four polypeptides
[5–8]. The largest of these polypeptides is the 70-kDa FAD-
containing flavoprotein subunit (Fp). The catalytic portion
of complex II is relatively hydrophilic and is formed by Fp
and an  30-kDa iron–sulfur protein subunit (Ip) that
contains three different types of iron-sulfur clusters. In
QFR, this region acts as a fumarate reductase (FRD),
catalyzing electron transfer from water soluble electron
donors, such as reduced FMN, to fumarate, while in SQR,
this region acts as a succinate dehydrogenase (SDH),

catalyzing oxidation of succinate by water-soluble electron
acceptors, such as phenazine methosulfate (PMS). Small
hydrophobic subunits, SdhC/FrdC or CybL ( 15 kDa),
and SdhD/FrdD or CybS ( 13 kDa) anchor this catalytic
portion to the membrane, and are required for electron
transfer between complex II and quinones (reviewed in [9]).
While the primary structures of the soluble catalytic
subunits are highly conserved between species, the sequence
and cofactor composition of the membrane anchor subunits
vary. Based on their b-heme composition, membrane
anchor domains have been grouped into three classes
[7]. Type A SQR/QFRs contain two b-hemes ligated to
four conserved histidine residues and include QFR from
Correspondence to K. Kita, Department of Biomedical Chemistry,
Graduate School of Medicine, The University of Tokyo,
7-3-1 Hongo, Bunkyo-ku, Tokyo 113–0033, Japan.
Fax: + 81 3 5841 3444, Tel.: + 81 3 5841 3526,
E-mail:
Abbreviations: QFR, quinol-fumarate reductase; SQR, succinate-
ubiquinone reductase; Fp, flavoprotein subunit; Ip, iron–sulfur
protein subunit; FRD, fumarate reductase; PMS, phenazine
methosulfate; SDH, succinate dehydrogenase; MK, menaquinone;
RQ, rhodoquinone; UQ, ubiquinone; MK-QFR, menaquinol-fuma-
rate reductase; RQ-QFR, rhodoquinol-fumarate reductase; C12E9,
polyoxyethylene-9-lauryl ether; DB, 2,3-dimethoxy-5-methyl-6-decyl-
1,4-benzoquinone; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-
2,4-tetrazolium bromide; DMQ, demethoxy ubiquinone.
Enzymes: succinate-quinone oxidoreductase (EC 1.3.5.1).
*Present address: Department of Biology, McGill University,
Montreal, Quebec, Canada.

(Received 18 November 2002, revised 23 February 2003,
accepted 3 March 2003)
Eur. J. Biochem. 270, 1863–1874 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03553.x
Wolinella succinogenes. Type B complex II contains one
b-heme, and this type includes most of the SQRs found
in aerobic bacteria and mitochondria. Type C complex II,
such as Escherichia coli QFR, contains no heme. While
bacterial QFRs are either type A or C, their mitochondrial
counterpart, QFR from adult Ascaris suum,istypeB[3].
The mechanisms of substrate binding and intramolecular
electron transfer in complex II have been elucidated by
crystallographic analyses of bacterial QFRs from W. suc-
cinogenes (type A) and E. coli (type C) [10–12]. Most
bacterial QFRs investigated to date use the low-potential
naphthoquinone menaquinone (MK, E
m
¢ , ) 74 mV) as an
electron donor to reduce fumarate (menaquinol-fumarate
reductase, MK-QFR) [13]. However, QFRs in mitochon-
dria use rhodoquinone (RQ), a low-potential benzoquinone
(E
m
¢ , ) 63 mV) [14] as a substrate for fumarate reduction
(rhodoquinol-fumarate reductase, RQ-QFR) [2,3]. RQ is
structurally similar to ubiquinone (UQ), but has an amino
group where UQ has a methoxy group (Fig. 1).
RQ-QFR has been identified in various eukaryote species,
including parasitic organisms such as the nematode A. suum
[3], the trematode, Schistosoma mansoni [15] and the cestode,
Hymenolepis nana [16], as well as in lower marine eukaryotes

[17]. Although RQ was identified originally in the purple
nonsulfur bacteria, Rhodospirillum rubrum [18], and has been
found in several species of bacteria [19–21], RQ-QFR has
never been reported in bacteria. However, several lines of
evidence suggest that complex II from the phototrophic
purple bacterium, Rhodoferax fermentans may be able to
catalyze the reduction of fumarate using reduced RQ as an
electron donor [22]. Firstly, both RQ and UQ are present in
R. fermentans as major quinone species, but MK is not
present [19]. Secondly, R. fermentans exhibits anaerobic
growth in the dark that is stimulated by bicarbonate, and
succinate is formed as the major end product, in addition to
formate, acetate, and lactate [23]. In fact, the membrane
fraction of R. fermentans exhibits fumarate reductase acti-
vity if reduced FMN is provided as an artificial electron
donor [24]. Thirdly, the RQ content of R. fermentans
increases under anaerobic conditions [22]. This suggests that
RQ functions as an electron carrier in anaerobic fumarate
respiration in R. fermentans.
For these reasons, we utilized R. fermentans to investigate
whether RQ functions as the electron donor for the QFR
activity of bacterial complex II, and if so, to determine the
evolutionary relationship between bacterial and mitochond-
rial complex II that functions as an RQ-QFR. We purified
complex II from anaerobically cultured R. fermentans and
showed that the enzyme catalyzes the reduction of fumarate
by reduced RQ. In addition, we cloned the R. fermentans
complex II genes in order to determine the structural
similarity with SQR, bacterial MK-QFR and mitochondrial
RQ-QFR. Biochemical and structural analyses revealed that

R. fermentans possesses type B complex II, and there is a
close evolutionary relationship with bacterial SQR.
Experimental Procedures
Bacterial strain and culture conditions
The phototrophic purple bacterium R. fermentans,strain
FR2 [22], was precultured in MYS
1
medium containing
20 m
M
sodium malate and 7.6 m
M
ammonium sulfate [24].
Preculture was performed anaerobically at 30 °C, under
incandescent illumination ( 5000 lx). Cells were then
diluted to 1 : 100 in FCYS medium
2
[23] containing
20 m
M
fructose and 30 m
M
sodium bicarbonate. Cells were
grown subsequently at 30 °C in full screw-capped bottles
under anaerobic conditions in the dark.
Preparation of membranes
All steps were performed at 4 °C. To obtain the membranes,
cells (10 g wet-weight) were suspended in 80 mL of 30 m
M
Tris/HCl (pH 8.0), containing 20 m

M
sucrose and 1 m
M
phenylmethylsulfonyl fluoride. Cells were treated with
5m
M
EDTA and 50 lgÆmL
)1
lysozyme for 10 min, and
then ruptured by French press (Ohtake, Tokyo) at
1500 kgÆcm
)2
. Unbroken cells and cell debris were removed
by centrifugation at 8000 g for 5 min, and the supernatant
was further centrifuged at 170 000 g for 60 min. After
washing with 30 m
M
Tris/HCl (pH 8.0), the membrane
component was centrifuged at 170 000 g for 60 min, and
the pellet was suspended in 30 m
M
Tris/HCl (pH 8.0)
containing 0.4
M
sucrose. This membrane suspension was
stored at )80 °C until use.
Purification of complex II
All steps were performed at 4 °C. To solubilize complex II,
crude membranes (100 mg protein) were suspended at
1mgÆmL

)1
protein in 10 m
M
Tris/HCl (pH 7.5), 1% (w/v)
polyoxyethylene-9-lauryl ether (C12E9) (Sigma) and 1 m
M
phenylmethylsulfonyl fluoride. This mixture was stirred for
60 min and then centrifuged at 170 000 g for 60 min. The
supernatant was applied to a column of DEAE-Cellulofine
(2 · 6 cm) (Seikagaku Kogyo, Tokyo) equilibrated with
10 m
M
Tris/HCl (pH 7.5), 0.1% (w/v) C12E9 and 5% (w/v)
Fig. 1. Quinone structures. RQ is structurally
similar to UQ, but retains a low mid-point
potential that is comparable to that of MK.
1864 H. Miyadera et al. (Eur. J. Biochem. 270) Ó FEBS 2003
sucrose. After washing with 40 mL of equilibration buffer
and 80 mL of equilibration buffer containing 50 m
M
NaCl,
complex II was eluted with 120 mL of equilibration buffer
containing a linear gradient of 50–150 m
M
NaCl at flow rate
of 14 mLÆh
)1
. Absorbance of each fraction was measured at
280 nm and 412 nm to determine protein and heme
concentration, respectively. Both SQR and RQ-QFR acti-

vities were monitored during elution. Purification by DEAE-
chromatography was conducted five times, each starting
with 100 mg protein ofmembranes. Peak fractions (fractions
168–178 in Fig. 2A) from each column were combined and
concentrated by centriplus-100 (Amicon, Inc.), and were
then applied to a column of Sephacryl S-300 H (1.5 · 70 cm)
(Pharmacia Biotech AB) equilibrated with 10 m
M
Tris/HCl
(pH 7.5), 0.1% (w/v) C12E9 and 5% (w/v) sucrose. Elution
was performed with the same buffer at a flow rate of
3.3 mLÆh
)1
, and 0.5-mL fractions were collected. Absorb-
ance at 280 nm and 412 nm, as well as RQ-QFR, SQR and
SDH activities were monitored during elution.
Synthesis of 3-amino-2-methoxy-5-methyl-6-
n
-decyl-
1,4-benzoquinone (
n
-decylrhodoquinone)
3-Hydroxy-2-methoxy-5-methyl-6-n-decyl-1,4-benzoquinone,
which was prepared using a previously reported method
[25], was mesylated with methanesulfonyl chloride in dry
tetrahydrofuran (THF) in the presence of triethylamine
3
at )20 °C (95% yield). The mesylate was reacted with
NaN
3

(2) in dry methanol at room temperature to produce
3-azido-2-methoxy-5-methyl-6-n-decyl-1,4-benzoquinone
(55% yield). Reduction of the azido compound with
NaBH
4
in methanol/0.1
M
4
NaOH (7 : 2) under argon gas
at room temperature produced crude n-decylrhodoquinol.
After oxidation of n-decylrhodoquinol to n-decylrhodo-
quinone with Ag
2
O in ethyl acetate, the final product was
purified by silica gel column chromatography (hexane/
CHCL
3
/AcOEt,70:25:5,82%yield).
1
HNMR(CDCl
3
,
300 MHz) d 0.88 (t, J ¼ 6.7 Hz, 3H, CH
3
), 1.2–1.5 (m, 16H,
-CH
2
-),1.97(s,3H,ArCH
3
),2.45(t,J ¼ 7.3 Hz, 2H,

ArCH
2
-),3.87(s,3H,OCH
3
),4.67(brs,2H,NH
2
).ESIMS
(m/z) 308.3 [M + H]
+
.
Measurement of enzymatic activities
All assays were performed at 30 °Cin50m
M
phosphate
buffer (pH 7.5) containing 0.05% (w/v) sucrose mono-
laurate. SQR activity was measured as described previously
[26] using 100 l
M
2,3-dimethoxy-5-methyl-6-n-decyl-1,4-
benzoquinone (DB) (Sigma) as the substrate. SDH
activity was measured by monitoring the change in
absorbance of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-
2,4-tetrazolium bromide (MTT) at 570 nm in the
Fig. 2. Chromatography of RQ-QFR. Elution
profile of R. fermentans complex II from
ion-exchange (A) and gel-filtration (B) column
chromatography. (d), absorbance at 280 nm;
(j), absorbance at 412 nm; ( m), RQ-QFR
activity; (s), SQR activity; (h), SDH activity.
(A) Membranes from the anaerobically cul-

tured R. fermentans were solubilized with 1%
(w/v) C12E9 in the presence of 1 m
M
phenyl-
methanesulfonyl fluoride. The supernatant
was applied to a column (2 · 6cm)equili-
brated with 10 m
M
Tris/HCl (pH 7.5) buffer
containing 0.1% (w/v) C12E9 and 5% (w/v)
sucrose. After washing with 50 m
M
NaCl in
the same buffer, complex II was eluted with
120 mL of the same buffer containing a linear
gradient of 50–150 m
M
NaCl at a flow rate of
14 mLÆh
)1
and 1 mL fractions were collected.
(B) Peak fractions from DEAE-Cellulofine
(fraction no. 168–178 in Fig. 2A) were pooled
and concentrated before being applied to
Sephacryl S-300 H (1.5 · 70 cm), equilibrated
with 10 m
M
Tris/HCl (pH 7.5) buffer con-
taining 0.1% (w/v) C12E9 and 5% (w/v)
sucrose.Elutionwasperformedinthesame

bufferataflowrateof3.3 mLÆh
)1
and 500 lL
fractions were collected.
Ó FEBS 2003 Complex II from Rhodoferax fermentans (Eur. J. Biochem. 270) 1865
presence of PMS [27], using an extinction coeffi-
cient of 17 m
M
)1
Æcm
)1
for MTT. To measure RQ-QFR
activity, synthetic n-decylrhodoquinone (100 l
M
) was used
as substrate [4]. RQ-QFR activity was measured anaero-
bically in a rubber-stopped cuvette containing 10 m
M
glucose, glucose oxidase (100 lgÆmL
)1
) (Boehringer Mann-
heim) and catalase (2 lgÆmL
)1
) (Wako) [28] and N
2
-trea-
ted buffer. RQ was reduced by 1 m
M
sodium borohydride
before each assay, and the enzymatic reaction was initiated

by addition of 5 m
M
sodium fumarate. Activity was meas-
ured by monitoring the change in absorbance of RQ at
283 nm, using an extinction coefficient of 7.9 m
M
)1
Æcm
)1
[19].
N-terminal sequence analysis
The purified complex II was electrophoresed on a 12.5%
polyacrylamide gel containing SDS, and electroblotted
onto a polyvinylidine difluoride membrane (Immobilon-
P
SQ
Millipore) using CAPS (3-[cyclohexylamino-1-pro-
panesulfonic acid]) buffer [29]. Protein bands were excised
and subjected to N-terminal sequence analysis. The
sequences were determined by automated Edman degra-
dation by using a Hewlett Packard G1005A protein
sequencer for reaction, and a Hewlett Packard HPLC
1090 for detection.
Cloning of genes encoding
R. fermentans
complex II
DNA isolation was conducted as described previously
[30]. To clone the gene encoding the Fp subunit, PCR
degenerate primers A and B were designed from the
conserved regions among known bacterial Fp subunits.

The primer sequences were as follows: primer A, 5¢-
CA(CT) GGN GCN AA(CT) CGN (CT)TN GG-3¢;
primer B, 5¢-(AG)TG NGC (AG)CC NCG NGA
(CT)TC-3¢, where N indicates either A, G, C or T.
Primer locations are shown in Figs 5, 6A. The primers
(0.2 l
M
each) and genomic DNA were combined with
5 lLof10· Taq polymerase buffer (Boehringer Mann-
heim), 200 l
M
of dNTP, and 2 l
M
of magnesium
chloride. The volume was brought up to 50 lLwith
distilled water, and 2.5 U of Taq DNA polymerase
(Boehringer Mannheim) were added. Each amplification
cycle consisted of DNA denaturation at 95 °Cfor
0.5 min, primer annealing at 50 °C for 0.5 min, and
extension at 72 °C for 1 min. After 30 cycles, a fragment
of the expected size (430 bp) was cloned into the plasmid
vector from a TA cloning kit (Invitrogen) and sequenced.
This fragment was then used as probe in subsequent
screening. A genomic library of R. fermentans was
constructed by inserting BglII fragments of R. fermentans
genomic DNA into kFIX II vector (Stratagene), according
to the manufacturer’s instructions. Plaque hybridization
was performed by the use of a DIG labeling and detection
system (Boehringer Mannheim). The positive clones
obtained from the library were subcloned into pUC119

to determine the sequences. Sequence analysis was
performed using a SHIMADZU DSQ-1000 sequencer
(Shimadzu) with a Thermo Sequenase fluorescent-labeled
primer cycle sequencing kit and 7-deaza-dGTP (Amer-
sham).
Phylogenetic analysis
Phylogenetic analysis was performed using the deduced
amino acid sequences of the complex II Fp subunit from
various species. The sequences were aligned by
CLUSTAL X
,
version 1.64b [31], and positions devoid of gaps (509
positions) were used for the subsequent phylogenetic
analysis. Analysis was performed by the maximum-likeli-
hood method of protein phylogeny [32] using
PROTML
version 2.3 [33]. Phylogenetic tree construction was also
performed by the neighbor-joining method using
CLUSTAL X
version 1.64b [31].
Other methods
Protein concentration was determined using the Lowry
method [34] with bovine serum albumin as a standard. SDS/
PAGE was carried out using a linear gradient gel (10% –
20%) (Biocraft, Tokyo). Absorption spectra were recorded
on a SHIMADZU UV-3000 dual wavelength spectrophoto-
meter (Shimadzu). Heme content was determined based on
the absorbance spectra of pyridine hemochromogen, as
described previously [35].
Results and Discussion

RQ-QFR activity of
R. fermentans
membranes
In order to study the kinetic properties of RQ-QFRs from
various species, we established previously a spectrophoto-
metric enzyme assay system that employs chemically
synthesized RQ having a short-side chain [4]. Using this
assay system, the RQ-QFR activity of membranes isolated
from anaerobically grown cells was examined because the
membrane of anaerobic cultured R. fermentans contains a
significant amount of RQ [22] and exhibits FRD activity
using reduced FMN as electron donor [24]. RQ-QFR
activity in the membrane fraction of anaerobically cultured
R. fermentans (39 nmolÆmin
)1
Æmg
)1
protein) was much
higher than that in the mitochondria of the adult parasitic
nematode A. suum (26 nmolÆmin
)1
Æmg
)1
protein). This
finding indicates that RQ functions as an electron donor
for fumarate reduction in R. fermentans.
Purification of complex II from
R. fermentans
Complex II was solubilized from the crude membrane using
the nonionic detergent, C12E9. Among several nonionic

detergents, C12E9 gave the best yield and highest specific
activity (data not shown), although both RQ-QFR and
SQR activities were decreased slightly upon extraction
(Table 1). Complex II was eluted from the DEAE-Cellulo-
fine column after washing the column with a buffer
containing 50 m
M
NaCl (Fig. 2A). Elution increased the
specific activities of RQ-QFR and SQR by 8-fold and
11-fold, respectively (Table 1). Because samples purified
using a large column showed lower specific activity, the peak
fractions obtained in five independent ion-exchange chro-
matography procedures were combined and applied to a
Sephacryl S-300 H gel-filtration column.
Further purification by gel-filtration chromatography
(Fig. 2B) resulted a 1.6-fold increase in RQ-QFR specific
1866 H. Miyadera et al. (Eur. J. Biochem. 270) Ó FEBS 2003
activity to 584 nmol min
)1
Æmg
)1
, and a 2.8-fold increase in
SQR specific activity to 4016 nmolÆmin
)1
Æmg
)1
(Table 1).
Coomassie-staining of SDS/PAGE revealed an approxi-
mate twofold increase in purity after gel-filtration chroma-
tography (Fig. 3). Therefore, the greater increase of SQR

specific activity, in comparison with that of RQ-QFR, may
be due to the loss of RQ-QFR activity during purification.
RQ-QFR activity was also observed to be more labile
during extraction and ion-exchange chromatography
(Table 1). Interestingly, when SDH activity was monitored
in the fractions eluted from the gel filtration column, two
separate peaks (peak I and peak II in Fig. 2B) were
observed. The absence of quinone-mediated electron trans-
fer in peak II fractions, along with SDS/PAGE results
indicated that peak II fractions did not contain the anchor
subunits, CybL and CybS (data not shown). This observa-
tion indicates that a portion of the enzyme complex
dissociated into catalytic and membrane anchor domains
during purification.
Subunit composition and properties of
R. fermentans
complex II
Figure 3 shows the polypeptide composition of samples
from each purification step. The purified R. fermentans
complex II (lane d) consists of four major polypeptides with
apparent molecular masses of 64.0, 28.6, 18.7 and 17.5 kDa
which correspond to the respective sizes of the Fp, Ip, CybL
and CybS subunits. The low
5
intensity of the CybS subunit
band may be due to its low binding affinity with Coomassie
blue dyes, as observed for E. coli SQR [36]. The purity of
the isolated RQ-QFR, as evaluated from SDS/PAGE
results, was approximately 75%.
Figure 4 shows the oxidized minus reduced spectra of the

purified enzyme. At room temperature, the spectra exhibits
an a-band at 556 nm with a shoulder at 550 nm and a
c-band at 424 nm. These properties are characteristic of a
b-type cytochrome. The purified enzyme contains a substoi-
chiometric amount of heme (1.3 nmol hemeÆmg
)1
protein),
as observed in purified complex II from other sources
[37–39].
Table 2 shows a comparison of the kinetic properties of
the purified enzyme and those of enzymes that utilize
benzoquinone as an electron donor or acceptor, such as
A. suum RQ-QFR and E. coli SQR. Using rhodoquinol
as the substrate for a QFR reaction, we found that
purified E. coli SQR shows a specific RQ-QFR activity in
the same range as the ubiquinol-fumarate reductase
activity reported by Maklashina et al.[28](Table2).
Compared with these QFR activities, R. fermentans com-
plex II shows a significantly higher RQ-QFR activity, and
this is even higher than that of A. suum RQ-QFR. This
finding is consistent with the fact that like A. suum RQ-
QFR, R. fermentans complex II has low K
m
values for
rhodoquinol and fumarate. R. fermentans complex II also
displays high SQR activity, and its kinetic parameters for
UQ and succinate are similar to the values observed for
E. coli SQR. This indicates that R. fermentans complex II
might function as SQR under aerobic conditions. In fact,
a study on the respiratory electron transfer pathway in

Table 1. Purification of complex II from anaerobically cultured R. fermentans.
Step Protein (mg)
Total activity
(nmolÆmin
)1
)
Specific activity
(nmolÆmin
)1
Æmg
)1
)
RQ-QFR SQR RQ-QFR (-fold) SQR (-fold)
Membranes 100.0 3939 10060 39 101
+ C12E9 100.0 2486 8655 25 (0.63) 86 (0.86)
C12E9 extract 45.0 2117 7616 47 (1.2) 169 (1.7)
DEAE-Cellulofine 6.4 2105 7299 328 (8.4) 1140 (11.3)
DEAE-Cellulofine
a
5.1 1877 7170 368 (9.4) 1405 (14)
Sephacryl S-300 H 1.7 970 6667 584 (15) 4016 (40)
a
Peak activity fractions from five separate DEAE-Cellulofine column chromatography procedures were pooled and applied to the Seph-
acryl S-300 H column (see Experimental Procedures).
Fig. 3. SDS/PAGE showing purification of R. fermentans complex II.
Lane (a), R. fermentans membrane (10 lg); lane (b) C12E9 extract
(8 lg); lane (c) complex II purified by DEAE-Cellulofine column
chromatography (10 lg); lane (d) complex II purified by Sephacryl
S-300 H (5 lg).
Ó FEBS 2003 Complex II from Rhodoferax fermentans (Eur. J. Biochem. 270) 1867

R. fermentans revealed that, under aerobic conditions,
complex II functions as SQR and participates in electron
transfer to high-potential iron-sulfur protein (HiPIP) via
complex III [40]. Finally, the ratio of RQ-QFR activity vs.
SQR activity (QFR/SQR in Table 2) was ten times higher
for R. fermentans complex II than for E. coli SQR,
indicating that the enzymatic properties of R. fermentans
complex II are clearly distinct from those of E. coli SQR.
This indicates that the RQ-QFR activity of R. fermentans
complex II is not simply the inverse reaction of bacterial
SQR, but that the enzyme may indeed function as an RQ-
QFR under dark, anaerobic conditions.
These results showed that R. fermentans complex II uses
reduced RQ as an electron donor for fumarate reduction,
and demonstrated for the first time that bacterial complex II
is able to function as an RQ-QFR. The enzymatic
properties, subunit composition, and heme content of
R. fermentans complex II indicate that, like A. suum
RQ-QFR, it is a type B complex II [7].
Primary structure of genes encoding
R. fermentans
RQ-QFR
In order to obtain information on the primary structure of
the enzyme, the genes encoding the four subunits of
R. fermentans complex II were cloned from the genomic
library of R. fermentans. The genes for the Fp, Ip, CybL,
and CybS subunits are designated as sdhA, sdhB, sdhC and
sdhD, respectively, based on the high similarity of the
deduced amino acid sequences with sdh genes from other
bacterial species. As shown in Fig. 5, the genes were found

inthesameorderastheE. coli sdh genes [41,42], and were
distinct from the E. coli frd genes, in which the genes for the
CybL and CybS subunits are located downstream of the
gene for the Ip subunit [43–45]. The deduced amino acid
sequences for CybL, CybS, Fp, and Ip subunits comprised
Fig. 4. Reduced-minus-oxidized difference spectra of R. fermentans
complex II at room temperature. The spectrum was recorded on a
Shimadzu UV-3000 spectrophotometer at 25 °C, with a light path
length of 10 mm. The sample (813 lgÆmL
)1
, fraction 112 in Fig. 2B) in
10 m
M
Tris/HCl (pH 7.5) buffer, containing 0.1% (w/v) C12E9 and
5% (w/v) sucrose was reduced with sodium dithionite. Spectrum A was
recorded between 400–600 nm, and spectrum B was recorded between
500–600 nm and amplified twofold.
Table 2. Kinetic properties of complex II. V
max
(lmolÆmin
)1
Æmg
)1
), representative data are shown. Almost identical values of V
max
and K
m
(standard error within 1–3%) were obtained in another preparation of each sample. RQH
2,
n-decyl RQ

;
UQ, n-decyl UQ.
Enzyme
RQ-QFR SQR
V
max
K
m
(l
M
)
V
max
K
m
(l
M
)
QFR/SQR
a
Fumarate RQH
2
Succinate UQ
R. fermentans complex II 1.17 35.7 39.9 7.30 262 6.2 0.160
A. suum RQ-QFR
b
0.89 16.6 40.6 1.39 625 11.4 0.642
E. coli SQR
c
0.22 74.6 60.6 13.30 277 4.8 0.016

a
V
max
for RQ-QFR/V
max
for SQR.
b
Purified from adult A. suum mitochondria [60],
c
Purified from E. coli GO103 [61].
Fig. 5. Organisation of genes encoding R. f ermentans complex II. Open
boxes represent sdhC, sdhD, sdhA and sdhB genes encoding the CybL,
CybS, Fp and Ip subunits, respectively, of the R. fermentans com-
plex II. Locations of the primers A and B are indicated by arrows.
Restriction enzyme sites used in cloning are also indicated: A, AccI; B,
BglII; E, EcoRI; H, HincII; S, SphI. The horizontal lines below the
restriction map indicate the subcloned fragments for sequencing.
1868 H. Miyadera et al. (Eur. J. Biochem. 270) Ó FEBS 2003
147, 121, 601, and 234 amino acids, respectively (GenBank
accession Nos BAA31213, BAA31214, BAA31215, and
BAA31216, respectively). In the case of Fp, Ip, and CybS
subunits, the deduced amino acid sequences contained a
region that was identical to the N-terminal sequence of the
purified enzyme (Fig. 6A,B,D). For the CybL subunit,
Fig. 6. Primary structure of R. fermentans complex II. (A) The nucleotide sequence and the deduced amino acid sequence of the R. fermentans sdhA
gene, encoding the Fp subunit. Numbers indicate the position of the amino acid from the first methionine. Asterisks denote the termination codon.
The N-terminal sequence obtained by Edman degradation of the purified enzyme is underlined. The circled residue indicates the FAD-binding
histidine [10,11]. The boxed residues indicate histidine and arginines in the active site [11]. A and B indicate the regions of primer A and primer B,
respectively. (B) Comparison of amino acid sequences for the three cysteine rich clusters of the Ip subunit. Conserved cysteine residues are boxed.
The numbers indicate the position of the amino acid from the first methionine, or from the N-terminal of mature enzymes in the case of

mitochondrial enzymes. The species names, references and GenBank accession numbers are as follows: EcMK-QFR, E. coli frdB [44] (AAC77113);
EcSQR, E. coli sdhB [42] (AAC73818); RfRQ-QFR, R. fermentans Ip (this work) (BAA31216); AsRQ-QFR, A. suum Ip [63] (BAA23716);
HsSQR, Homo sapiens Ip [67] (BAA01089). (C and D) Comparison of the amino acid sequences of the CybL subunit (C) and the CybS subunit (D)
of complex II. For mitochondrial enzymes, the sequences of the mature enzymes are shown. The numbers indicate the position of the amino acid
from the N-terminus. The amino acids identical to the sequences of R. fermentans complex II are boxed. For the CybS subunit, the N-terminal
sequences obtained by Edman degradation of the purified enzyme are underlined. Open arrows indicate the possible heme ligand histidines. The
species names, references, and GenBank accession numbers are as follows: in Fig. 6C: EcMK-QFR, E. coli frdC [45] (AAC77112); EcSQR, E. coli
sdhC [41] (CAA25485); RfRQ-QFR, R. fermentans sdhC (this work) (BAA31213); AsRQ-QFR, A. suum CybL [65] (BAA11232); HsSQR, Homo
sapiens CybL [68] (BAA31998). In Fig. 6D: EcMK-QFR, E. coli frdD [45] (AAC77111); EcSQR, E. coli sdhD [41] (CAA25486); RfRQ-QFR,
R. fermentans sdhD (this work) (BAA31214); AsRQ-QFR, A. suum CybS [64] (BAA11233); HsSQR, H. sapiens CybS [68] (BAA22054).
Ó FEBS 2003 Complex II from Rhodoferax fermentans (Eur. J. Biochem. 270) 1869
N-terminal sequence analysis was unsuccessful, possibly due
to N-terminal blockage.
In the sdhA gene, the FAD-binding histidine [10,11], the
regions for AMP binding [5]
6
,andtheactivesitehistidine
and arginines [11] were found in the appropriate corres-
ponding regions (Fig. 6A). In the sdhB gene, the three
cysteine-rich clusters are highly conserved with other
complex II proteins (Fig. 6B). The exception is the third
cysteine in the S-1 cluster, that is replaced by an aspartate, as
observed in E. coli SQR [42]. The same substitution in
E. coli MK-QFR by site-directed mutagenesis had no
deleterious effect on physical or enzymatic properties [46],
indicating that the S-1 cluster of R. fermentans complex II,
as well as that of E. coli SQR, may form a noncysteinyl,
oxygenic ligand for the [2Fe-2S] center.
Figure 6C,D show the deduced amino acid sequences for
the CybL and CybS subunits, for which the calculated

molecular masses are 16.5 and 13.9 kDa, respectively. As is
often observed with these two subunits of complex II [47],
the molecular masses determined from amino acid sequen-
ces are slightly lower than those estimated by SDS/PAGE
mobility (Fig. 3). Both subunits are predicted to form three
membrane-spanning regions by hydropathy plot [48] (data
not shown). The axial heme ligand histidines identified in
E. coli SQR [49,50] are conserved in the second transmem-
brane segments of each subunit, while the ligand histidines
for the low-spin heme identified in type A complex II, such
as W. succinogenes MK-QFR [11] and Bacillus subtilis SQR
[51], are not found in the corresponding regions. Together
with the results from the heme content analysis, these
findings indicate that R. fermentans complex II contains
one mol heme per enzyme, that is characteristic of type B
complex II, such as E. coli SQR [47] and A. suum RQ-
QFR [52].
Table 3 summarizes the amino acid sequence similarities
between each subunit of bacterial/mitochondrial QFR and
SQR. In all four subunits, R. fermentans complex II retains
significantly higher identity with bacterial SQR than with
bacterial MK-QFR. The similarity of R. fermentans RQ-
QFR to mitochondrial RQ-QFR is in the same range as its
similarity to mitochondrial SQR.
Evolution of bacterial RQ-QFR and quinones
The identification of complex II displaying a high RQ-QFR
activity in both bacteria and mitochondria prompted us to
analyze whether R. fermentans complexIIsharesacom-
mon evolutionary origin with mitochondrial RQ-QFR. A
phylogenetic tree was constructed based on the deduced

amino acid sequences of the Fp subunit (Fig. 7). Eubacterial
and mitochondrial complex II branch out into major three
groups with high bootstrap proportions. Group 1 contains
bacterial QFR and SQR, that possess a single membrane
anchor subunit. This group corresponds to type A com-
plex II. Bacterial MK-QFRs with two membrane anchor
subunits are located in Group 2. R. fermentans complex II
is found in Group 3, that consists of type B complex II.
These findings strongly suggest that R. fermentans com-
plex II evolved from bacterial SQR, and not directly
from MK-QFR. A. suum RQ-QFR comprises a subgroup
together with mitochondrial SQR, but is not directly linked
to R. fermentans complex II. This indicates that A. suum
RQ-QFR evolved from mitochondrial SQR, and is not
directly derived from endosymbiotic bacteria containing
RQ-QFR. From these results, it was concluded that
prokaryotic and eukaryotic RQ-QFR evolved independ-
ently from SQR, even though both enzymes catalyze the
same reaction.
The above result also implies that the transition of
quinone specificity from UQ to RQ occurred in both
bacteria and mitochondria in the course of the evolution of
complex II. It is believed that in the ancient anaerobic
environment, soluble fumarate reductase was used for
respiration in order to maintain the redox balance in the cell
[53]. When bacterial fumarate reductase became membrane-
bound, the reducing equivalents were possibly transferred
from MK, a process that occurs in the anaerobic respiratory
chain of present-day E. coli. As it evolved into aerobic SQR
in proteobacteria, a high-potential benzoquinone, UQ, was

employed for aerobic respiration, although a number of
bacteria, such as B. subtilis, use MK in succinate oxidation
[7]. The complex II found in present-day R. fermentans and
adult A. suum, that uses the low-potential benzoquinone
RQ as an electron donor for fumarate reduction, may have
then appeared. This indicates that RQ might have emerged
as a respiratory component compared more recently than
UQ. In fact, a phylogenetic tree of bacteria constructed
using 16s rRNA shows that RQ-containing species have
emerged from UQ-containing proteobacteria species [20].
The use of RQ in respiration might have enabled anaerobic
metabolism to develop in both bacteria and mitochondria.
The increase in RQ content under light, anaerobic condi-
tions [22] also suggests that R. fermentans complex II might
catalyze the RQ-QFR reaction in the anaerobic photosyn-
thetic electron transfer chain, and play a role in regulating
the redox balance during photosynthesis [54].
The mechanism by which evolutionarily unrelated
organisms commonly acquired RQ for use in anaerobic
respiration remains unknown. This question might be
answered partly by identifying the RQ biosynthesis pathway
in these phylogenetically diverse species. A study performed
on R. rubrum [55], Euglena glacilis [56] and Fasciola hepa-
tica [57] suggested that RQ is synthesized via the UQ
biosynthesis pathway and that UQ is a possible precursor in
RQ biosynthesis [55,56]. However, our recent study on the
nematode Caenorhabditis elegans, an organism that con-
tains RQ [58], showed that the UQ biosynthesis intermedi-
ate, demethoxy ubiquinone (DMQ), is accumulated in place
of UQ in the long-lived mutant clk-1 [26]. Importantly, the

clk-1 mutant contains RQ, despite the apparent absence of
UQ [59]. This indicates strongly that UQ may not be a
Table 3. Comparison of amino acid sequence similarities of complex II.
Enzyme
Amino acid sequence identity
to R. fermentans complex II
(%)
ReferencesFp Ip CybL CybS
E. coli MK-QFR 44.5 44.8 18.3 28.9 [43–45]
E. coli SQR 62.7 71.3 40.1 47.9 [41, 42]
A. suum RQ-QFR 55.7 63.2 19.0 24.8 [62–65]
H. sapiens SQR 57.5 62.0 26.5 24.0 [66–68]
1870 H. Miyadera et al. (Eur. J. Biochem. 270) Ó FEBS 2003
biosynthesis precursor of RQ in nematodes. Further study
on RQ biosynthesis pathways in RQ-containing pro/
eukaryotes is necessary to clarify how the anaerobic
respiratory chain may have evolved in bacteria and
mitochondria, thus facilitating their adaptation from aero-
bic to anoxic environments.
Fig. 7. Phylogenetic tree based on deduced amino acid sequences of various Fp subunits. The tree was constructed by the maximum-likelihood method
[32]. Horizontal length indicates the estimated number of substitutions per site. Local bootstrap values calculated by
PROTML
[33] are indicated on
the each node. Three major clusters are designated as Group 1, Group 2 and Group 3. Mitochondrial complex II proteins are boxed by a dotted
line. The physiological enzyme activities as well as the quinone species used by the respective enzymes (in parenthesis) are indicated. In the case of
C. trachomatis, H. influenzae, M. tuberculosis, S. frigidimarina,andA. aeolicus, the function of the enzyme is yet to be biochemically determined.
H. influenzae and M. tuberculosis are known to contain demethylmenaquinone (DMK) and MK, respectively [69]. References and GenBank
accession numbers for each sequence are as follows: C. trachomatis (AAC68194); B. subtilis [70] (P08065); P. macerans [71] (CAA69872);
W. succinogenes [72] (P17412); H. pylori [73] (AAC46064); P. vulgaris [74] (P20922); E. coli frdA [43] (AAC77114); H. influenzae (P44894);
M. tubercurosis frdA (Q10760); M. tubercurosis sdhA (CAA17090); S. frigidimarina (Y13760); E. coli sdhA [41] (AAC73817); C. burnetii [75]

(P51054); R. fermentans (this work) (BAA31215); P. denitrificans (Q59661); B. japonicum [76] (AAC17942); R. prowasekii [77] (P31038); S. cere-
visiae [78] (S34793); B. taurus [79] (AAA30758); H. sapiens [66] (BAA06332); A. suum [62] (BAA21636); C. elegans [62] (BAA21637); D. immitis
[80] (S78630); A. aeolicus frdA (AAC06812); S. acidocaldarius [81] (CAA70249).
Ó FEBS 2003 Complex II from Rhodoferax fermentans (Eur. J. Biochem. 270) 1871
Acknowledgements
This study was supported by a grant-in-aid for scientific research on
priority areas from the Ministry of Education, Science, Culture and
Sport, Japan (13226015 and 13854011) and for research on emerging
and re-emerging infectious diseases from the Ministry of Health and
Welfare. H. M. was supported by the Iwadare Foundation. We would
also like to acknowledge Dr T. Hashimoto, (Institute of Statistical
Mathematics) for helpful advice regarding phylogenetic analysis, and to
Dr T. Mogi, (The University of Tokyo) for the purified E. coli SQR.
References
1. Kro
¨
ger,A.,Geisler,V.,Lemma,E.,Theis,F.&Lenger,R.(1992)
Bacterial fumarate respiration. Arch. Microbiol. 158, 311–314.
2. Tielens, A.G.M. & Van Hellemond, J.J. (1998) The electron
transport chain in anaerobically functioning eukaryotes. Biochim.
Biophys. Acta 1365, 71–78.
3. Kita,K.,Hirawake,H.,Miyadera,H.,Amino,H.&Takeo,S.
(2002) Role of complex II in anaerobic respiration of the parasite
mitochondria from Ascaris suum and Plasmodium falciparum.
Biochim. Biophys. Acta 1553, 123–139.
4. Omura, T., Miyadera, H., Ui, H., Shiomi, K., Yamaguchi, Y.,
Masuma, R., Nagamitsu, T., Takano, D., Sunazuka, T., Harder,
A., Ko
¨
lbl, H., Namikoshi, M., Miyoshi, H., Sakamoto, K. & Kita,

K. (2001) An anthelmintic compound, nafuredin, shows selective
inhibition of complex I in helminth mitochondria. Proc. Natl
Acad. Sci. USA 98, 60–62.
5. Ackrell, B.A.C., Johnson, M.K., Gunsalus, R.P. & Cecchini, G.
(1992) Chemistry and Biochemistry of Flavoenzymes (Muller, F.,
eds), Vol. III, pp. 229–297. CRC Press, London.
6. Ha
¨
gerha
¨
ll, C. (1997) Succinate: quinone oxidoreductases. Varia-
tions on a conserved theme. Biochem. Biophys. Acta 1320,
107–141.
7. Ohnishi, T., Moser, C.C., Page, C.C., Dutton, P.L. & Yano, T.
(2000) Simple redox-linked proton-transfer design: new insights
from structures of quinol-fumarate reductase. Structure Fold. Dis.
8, R23–R32.
8. Lancaster, C.R.D. (2001) Succinate-quinone oxidoreductases-
what can we learn from Wolinella succinogenes quinol: fumarate
reductase? FEBS Lett. 504, 133–141.
9. Lancaster, C.R.D., ed. (2002) Special issue for complex II.
Biochim. Biophys. Acta 1553, 1–176.
10. Iverson, T.M., Luna-Chavez, C., Cecchini, G. & Rees, D.C. (1999)
Structure of the Escherichia coli fumarate reductase respiratory
complex. Science 284, 1961–1966.
11. Lancaster, C.R.D., Kro
¨
ger, A., Auer, M. & Michel, H. (1999)
Structure of fumarate reductase from Wolinella succinogenes at
2.2A

˚
resolution. Nature 402, 377–385.
12. Ackrell, B.A.C. (2000) Progress in understanding structure-func-
tion relationships in respiratory chain complex II. FEBS Lett. 466,
1–5.
13. Unden, G. & Bongaerts, J. (1997) Alternative respiratory path-
ways of Escherichia coli: energetics and transcriptional regulation
in response to electron acceptors. Biochim. Biophys. Acta 1320,
217–234.
14. Erabi, T., Higuti, T., Kakuno, T., Yamashita, J., Tanaka, M. &
Horio, T. (1975) Polarographic studies on ubiquinone-10 and
rhodoquinone bound with chromatophores from Rhodospirillum
rubrum. J. Biochem. 78, 795–801.
15. Van Hellemond, J.J., Van Remoortere, A. & Tielens, A.G.M. (1997)
Schistosoma mansoni sporocysts contain rhodoquinone and pro-
duce succinate by fumarate reduction. Parasitology 115, 177–182.
16. Fioravanti, C.F. & Kim, Y. (1988) Rhodoquinone requirement of
the Hymenolepis diminuta mitochondrial electron transport sys-
tem. Mol. Biochem. Parasitol. 28, 129–134.
17. Van Hellemond, J.J., Klockiewicz, M., Gaasenbeek, C.P.H.,
Roos, M.H. & Tielens, A.G.M. (1995) Rhodoquinone and com-
plex II of the electron transport chain in anaerobically functioning
eukaryotes. J. Biol. Chem. 270, 31065–31070.
18. Glover, J. & Therelfall, D.R. (1962) A new quinone (rhodoqui-
none) related to ubiquinone in the photosynthetic bacterium,
Rhodospirillum rubrum. Biochem. J. 85, 14P.
19. Hiraishi, A. & Hoshino, Y. (1984) Distribution of rhodoquinone
in Rhodospirillaceae and its taxonomic implications. J. Gen. Appl.
Microbiol. 30, 435–448.
20. Hiraishi, A., Shin, Y K. & Sugiyama, J. (1995) Brachymonas

denitrificans gen. nov., sp. nov., an aerobic chemoorganotrophic
bacterium which contains rhodoquinones, and evolutionary rela-
tions of rhodoquinone producers to bacterial species with various
quinone classes. J. Gen. Appl. Microbiol. 41, 99–117.
21. Hiraishi, A. & Ueda, Y. (1994) Rhodoplanes gen. nov., a new genus
of phototrophic bacteria including Rhodosseudomonas rosea as
Rhodoplanes roseus comb.nov. & Rhodoplanes elegans sp. nov. Int.
J. Sys. Bacteriol. 44, 665–673.
22. Hiraishi, A., Hoshino, Y. & Satoh, T. (1991) Rhodoferax fer-
mentans gen. nov., sp. nov., a phototrophic purple non-sulfur
bacterium previously referred to as the ÔRhodocyclus gelatinosus-
likeÕ group. Arch. Microbiol. 155, 330–336.
23. Hiraishi, A. (1988) Bicarbonate-stimulated dark fermentative
growth of a phototrophic purple non-sulfur bacterium. FEMS
Microbiol. Lett. 56, 199–202.
24. Hiraishi, A. (1988) Fumarate reduction systems in members of
the family Rhodospirillaceae with different quinone types. Arch.
Microbiol. 150, 56–60.
25. Ohshima, M., Miyoshi, H., Sakamoto, K., Takegami, K., Iwata,
J., Kuwabara, K., Iwamura, H. & Yagi, T. (1998) Characteriza-
tion of the ubiquinone reduction site of mitochondrial complex I
using bulky synthetic ubiquinones. Biochemistry 37, 6436–6445.
26. Miyadera, H., Amino, H., Hiraishi, A., Taka, H., Murayama, K.,
Miyoshi, H., Sakamoto, K., Ishii, N., Hekimi, S. & Kita, K. (2001)
Altered quinone biosynthesis in the long-lived clk-1 mutants of
Caenorhabditis elegans. J. Biol. Chem. 276, 7713–7716.
27. Futai, M. (1972) Membrane
D
-lactate dehydrogenase from
Escherichia coli. Purification and properties. Biochemistry 12,

2468–2474.
28. Maklashina, E. & Cecchini, G. (1999) Comparison of catalytic
activity and inhibitors of quinone reactions of succinate dehy-
drogenase (succinate-ubiquinone oxidoreductase) and fumarate
reductase (menaquinol-fumarate oxidoreductase) from Escheri-
chia coli. Arch. Biochem. Biophys. 369, 223–232.
29. Matsudaira, P. (1987) Sequence from picomole quantities of
proteins electroblotted onto polyvinylidene difluoride membranes.
J. Biol. Chem. 262, 10035–10038.
30. Sambrook, J., Fritsch, E.F. & Maniatis, T. (1989) Molecular
Cloning: a Laboratory Manual. Cold Spring Harbor Laboratory
Press, Cold Spring Harbor, New York.
31. Thompson, J.D., Gibson, T.J., Plewnlak, F., Jeanmougin, F. &
Higgins, D.G. (1997) The CLUSTAL–windows interface: flexible
strategies for multiple sequence alignment aided by quality ana-
lysis tools. Nucleic Acids Res. 24, 4876–4882.
32. Kishino, H., Miyata, T. & Hasegawa, M. (1990) Maximum
likelihood inference of protein phylogeny and the origin of
chloroplasts. J. Mol. Evol. 31, 151–160.
33. Adachi, J. & Hasegawa, M. (1996) Computer Science Monographs,
pp. 72–76. The Institute of Statistical Mathematics, Tokyo.
34. Lowry, O.H., Rosebrough, N.J., Farr, A.L. & Randall, R.J.
(1951) Protein measurement with the folin phenol reagent. J. Biol.
Chem. 193, 265–275.
35.Kita,K.,Yamato,I.&Anraku,Y.(1978)Purificationand
properties of cytochrome b
556
in the respiratory chain of aero-
bically grown Escherichia coli. J. Biol. Chem. 253, 8910–8915.
1872 H. Miyadera et al. (Eur. J. Biochem. 270) Ó FEBS 2003

36. Yang, X., Yu, L. & C A. (1997) The quinone-binding site in
succinate-ubiquinone reductase from Escherichia coli. Quinone-
binding domain and amino acid residues involved in quinone
binding. J. Biol. Chem. 272, 9683–9689.
37. Yu, L. & C A. (1982) Quantitative resolution of succinate-cyto-
chrome c reductase into succinate-ubiquinone and ubiquinol-
cytochrome c reductases. J. Biol. Chem. 257, 2016–2021.
38. Tushurashvili, P.R., Gavrikova, E.V., Ledenev, A.N. & Vino-
gradov, A.D. (1985) Studies on the succinate dehydrogenating
system. Isolation and properties of the mitochondrial succinate-
ubiquinone reductase. Biochim. Biophys. Acta 809, 145–159.
39. Pennoyer, J.D., Ohnishi, T. & Trumpower, B.L. (1988) Purifica-
tion and properties of succinate-ubiquinone oxidoreductase com-
plex from Paracoccus denitrificans. Biochim. Biophys. Acta 935,
195–207.
40. Hochkoeppler, A., Kofod, P. & Zannoni, D. (1995) HiPIP oxido-
reductase activity in membranes from aerobically grown cells of
the facultative phototroph Rhodoferax fermentans. FEBS Lett.
375, 197–200.
41. Wood, D., Darlison, M.G., Wilde, R.J. & Guest, J.R. (1984)
Nucleotide sequence encoding the flavoprotein and hydrophobic
subunits of the succinate dehydrogenase of Escherichia coli. Bio-
chem. J. 222, 519–534.
42. Darlison, M.G. & Guest, J.R. (1984) Nucleotide sequence
encoding the iron-sulphur protein subunit of the succinate
dehydrogenase of Escherichia coli. Biochem. J. 223, 507–517.
43. Cole, S.T. (1982) Nucleotide sequence coding for the flavoprotein
subunit of the fumarate reductase of Escherichia coli. Eur. J.
Biochem. 122, 479–484.
44. Cole, S.T., Grundstro

¨
m,T.,Jaulin,B.,Robinson,J.J.&Weiner,
J.H. (1982) Location and nucleotide sequence of frdB, the gene
coding for the iron-sulphur protein subunit of the fumarate
reductase of Escherichia coli. Eur. J. Biochem. 126, 211–216.
45. Grundstro
¨
m, T. & Jaulin, B. (1982) Overlap between ampC and
frd operons on the Escherichia coli chromosome. Proc.NatlAcad.
Sci. USA 79, 1111–1115.
46.Werth,M.T.,Sices,H.,Cecchini,G.,Schro
¨
der, I., Lasage, S.,
Gunsalus, R.P. & Johnson, M.K. (1992) Evidence for non-
cysteinyl coordination of the [2Fe-2S] cluster in Escherichia coli
succinate dehydrogenase. FEBS Lett. 299, 1–4.
47. Kita, K., Vibat, C.R.T., Meinhardt, S., Guest, J.R. & Gennis,
R.B. (1989) One-step purification from Escherichia coli of complex
II associated with succinate-reducible cytochrome b
556
. J. Biol.
Chem. 264, 2672–2677.
48. Kyte, J. & Doolittle, R.F. (1982) A simple method for displaying
the hydropathic character of a protein. J. Mol. Biol. 157, 105–132.
49. Vivat, C.R.T., Cecchini, G., Nakamura, K., Kita, K. & Gennis,
R.B. (1998) Localization of histidine residues responsible for heme
axial ligation in cytochrome b
556
of complex II in Escherichia coli.
Biochemistry 37, 4148–4159.

50. Maklashina, E., Rothery, R.A., Weiner, J.H. & Cecchini, G.
(2001) Retention of heme in axial ligand mutants of succinate-
ubiquinone oxidoreductase (complex II) from Escherichia coli.
J. Biol. Chem. 276, 18968–18976.
51. Ha
¨
gerha
¨
ll, C., Friden, H., Aasa, R. & Hederstedt, L. (1995)
Transmembrane topology and axial ligands to hemes in the
cytochrome b subunit of Bacillus subtilis succinate: menaquinone
reductase. Biochemistry 34, 11080–11089.
52. Takamiya, S., Furushima, R. & Oya, H. (1986) Electron-transfer
complexes of Ascaris suum muscle mitochondria. II. Succinate-
coenzyme Q reductase (complex II) associated with substrate-
reducible cytochrome b
558
. Biochim. Biophys. Acta 848, 99–107.
53. Hederstedt, L. (1999) Respiration without O
2
. Science 284, 1941–
1942.
54. Ferguson, S.J., Jackson, J.B. & McEwan, A.G. (1987) Anaerobic
respiration in the Rhodospirillaceae: characterization of pathways
and evaluation of roles in redox balancing during photosynthesis.
FEMS Micorobiol. Rev. 46, 117–143.
55. Parson, W.W. & Rudney, H. (1965) The biosynthesis of ubiqui-
none and rhodoquinone from p-hydroxybenzoate and p-hydro-
xybenzaldehyde in Rhodospirillum rubrum. J. Biol. Chem. 240,
1855–1863.

56. Powls, R. & Hemming, F.W. (1966) The biosynthesis of quinones
from p-hydroxybenzonic acid in Euglena gracilis var. bacillaris.
Phytochemistry 5, 1249–1255.
57. Van Hellemond, J.J., Luijten, M., Fiesch, F.M., Gaasenbeek, C.P.
& Tielens, A.G.M. (1996) Rhodoquinone is synthesized de novo by
Fasciola hepatica. Mol. Biochem. Parasitol. 82, 217–226.
58. Takamiya, S., Matsui, T., Taka, H., Murayama, K., Matsuda, M.
& Aoki, T. (1999) Free-living nematodes Caenorhabditis elegans
possess in their mitochondria an additional rhodoquinone, an
essential component of the eukaryotic fumarate reductase system.
Arch. Biochem. Biophys. 371, 284–289.
59. Jonassen, T., Larsen, P.L. & Clarke, C.F. (2001) A dietary source
of coenzyme Q is essential for growth of long-lived Caenorhabditis
elegans clk-1 mutants. Proc. Natl Acad. Sci. USA 98, 421–426.
60. Saruta, F., Kuramochi, T., Nakamura, K. & Takamiya, S., Yu,
Y., Aoki, T., Sekimizu, K., Kojima, S. & Kita, K. (1995) Stage-
specific isoforms of complex II in mitochondria from the parasitic
nematode, Ascaris suum. J. Biol. Chem. 270, 928–932.
61. Oden, K.L., DeVeaux, L.C., Vibat, C.R.T., Cronan, J.E. &
Gennis, R.B. (1990) Genomic replacement in Escherichia coli K-12
using covalently closed circular plasmid DNA. Gene 96, 29–36.
62. Kuramochi, T., Hirawake, H., Kojima, S., Takamiya, S.,
Furushima, R., Aoki, T., Komuniecki, R. & Kita, K. (1994)
Sequence comparison between the flavoprotein subunit of the
fumarate reductase (complex II) of the anaerobic parasitic
nematode, Ascaris suum and the succinate dehydrogenase of the
aerobic, free-living nematode, Caenorhabditis elegans. Mol.
Biochem. Parasitol. 68, 177–187.
63.Amino,H.,Wang,H.,Hirawake,H.,Saruta,F.,Mizuchi,D.,
Mineki, R., Shindo, N., Murayama, K., Takamiya, S., Aoki, T.,

Kojima, S. & Kita, K. (2000) Stage-specific isoforms of Ascaris
suum complex II: the fumarate reductase of the parasitic adult and
the succinate dehydrogenase of free-living larvae share a common
iron-sulfur subunit. Mol. Biochem. Parasitol. 106, 63–76.
64. Saruta, F., Hirawake, H., Takamiya, S., Ma, Y C., Aoki, T.,
Sekimizu, K., Kojima, S. & Kita, K. (1996) Cloning of a cDNA
encoding the small subunit of cytochrome b
558
(cybS) of
mitochondrial fumarate reductase (complex II) from adult Ascaris
suum. Biochim. Biophys. Acta 1276, 1–5.
65. Kita, K., Hirawake, H. & Takamiya, S. (1997) Cytochromes in the
respiratory chain of helminth mitochondria. Int. J. Parasitol. 27,
617–630.
66. Hirawake, H., Wang, H., Kuramochi, T., Kojima, S. & Kita, K.
(1994) Human complex II (succinate-ubiquinone oxidoreductase):
cDNA cloning of the flavoprotein (Fp) subunit of liver mito-
chondria. J. Biochem. 116, 221–227.
67. Kita, K., Oya, H., Gennis, R.B., Ackrell, B.A.C. & Kasahara, M.
(1990) Human complex II (succinate-ubiquinone oxidoreductase):
cDNA cloning of iron sulfur (Ip) subunit of liver mitochondria.
Biochem. Biophys. Res. Commun. 166, 101–108.
68. Hirawake,H.,Taniwaki,M.,Tamura,A.,Kojima,S.&Kita,K.
(1997) Cytochrome b in human complex II: cDNA cloning of the
components in liver mitochondria and chromosome assignment of
the genes for the large (SDHC) and small (SDHD) subunits to
1q21 and 11q23. Cytogenet. Cell Genet. 79, 132–138.
69. Collins, M.T. & Jones, D. (1981) Distribution of isoprenoid qui-
none structural types in bacteria and their taxonomic implications.
Microbiol. Rev. 45, 316–354.

70. Phillips, M.K., Hederstedt, L., Hasnain, S., Rutberg, L. & Guest,
J.R. (1987) Nucleotide sequence encoding the flavoprotein and
Ó FEBS 2003 Complex II from Rhodoferax fermentans (Eur. J. Biochem. 270) 1873
iron-sulfur protein subunits of the Bacillus subtilis PY79 succinate
dehydrogenase complex. J. Bacteriol. 169, 864–873.
71. Schirawski, J., Hankeln, T. & Unden, G. (1998) Expression of the
succinate dehydrogenase genes (sdhCAB) from the facultatively
anaerobic Paenibacillus macerans during aerobic growth. Arch.
Microbiol. 170, 304–308.
72. Lauterbach, F., Ko
¨
rtner,C.,Albracht,S.P.J.,Unden,G.&
Kro
¨
ger, A. (1990) The fumarate reductase operon of Wolinella
succinogenes. Arch. Microbiol. 154, 386–393.
73. Ge, Z., Jiang, Q., Kalisiak, M.S. & Taylor, D.E. (1997) Cloning
and functional characterization of Helicobacter pylori fumarate
reductase operon comprising three structural genes coding for
subunits C, A and B. Gene 204, 227–234.
74. Cole, S.T. (1987) Nucleotide sequence and comparative analysis
of the frd operon encoding the fumarate reductase of Proteus
vulgaris. Eur. J. Biochem. 167, 481–488.
75. Heinzen, R.A., Mo, Y Y., Robertson, S.J. & Mallavia, L.P.
(1995) Characterization of the succinate dehydrogenase-encoding
gene cluster (sdh) from the rickettsia Coxiella burnetii. Gene 155,
27–34.
76. Westenberg,D.J.&Guerinot,M.L.(1999)Succinatedehydro-
genase (Sdh) from Bradyrhizobium japonicum is closely related to
mitochondrial Sdh. J. Bacteriol. 181, 4676–4679.

77. Aliabadi, Z., Winkler, H.H. & Wood, D.O. (1993) Isolation and
characterization of the Rickettsia prowazekii gene encoding
the flavoprotein subunit of succinate dehydrogenase. Gene 133,
135–140.
78. Robinson, K.M. & Lemire, B.D. (1992) Isolation and nucleotide
sequence of the Saccharomyces cerevisiae gene for the succinate
dehydrogenase flavoprotein subunit. J. Biol. Chem. 267, 10101–
10107.
79. Birch-Machin, M.A., Farnsworth, L., Ackrell, B.A.C., Cochran,
B., Jackson, S., Bindoff, L.A., Aitken, A., Diamond, A.G. &
Turnbull, D.M. (1992) The sequence of the flavoprotein subunit of
bovine heart succinate dehydrogenase. J. Biol. Chem. 267, 11553–
11558.
80. Kuramochi, T., Kita, K., Takamiya, S., Kojima, S. & Hayasaki,
M. (1995) Comparative study and cDNA cloning of the
flavoprotein subunit of mitochondrial complex II from the dog
heartworm, Dirofilaria immitis. Comp. Biochem. Physiol. 111B,
491–502.
81. Janssen, S., Scha
¨
fer, G., Anemu
¨
ller, S. & Moll, R. (1997) A suc-
cinate dehydrogenase with novel structure and properties from the
hyperthermophilic archaeon Sulfolobus acidocaldarius: genetic and
biophysical characterization. J. Bacteriol. 179, 5560–5569.
1874 H. Miyadera et al. (Eur. J. Biochem. 270) Ó FEBS 2003