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 R
Trang 1Complex II from phototrophic purple bacterium Rhodoferax
Hiroko Miyadera1,*, Akira Hiraishi2, Hideto Miyoshi3, Kimitoshi Sakamoto3, Reiko Mineki4,
Kimie Murayama4, Kenji V P Nagashima5, Katsumi Matsuura5, Somei Kojima6and Kiyoshi Kita1
1
Department of Biomedical Chemistry, Graduate School of Medicine, The University of Tokyo, Japan;2Department of Ecological Engineering, Toyohashi University of Technology, Japan;3Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Japan;4Division of Biochemical Analysis, Central Laboratory of Medical Science, Juntendo University
School of Medicine, Tokyo, Japan;5Department 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: kitak@m.u-tokyo.ac.jp
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-fumamenaquinol-fuma-rate 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)
Trang 2Wolinella 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, is type B [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, Em¢ ,) 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
(Em¢ ,) 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 fermentanspossesses 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
20 mMsodium malate and 7.6 mMammonium sulfate [24] Preculture was performed anaerobically at 30C, under incandescent illumination ( 5000 lx) Cells were then diluted to 1 : 100 in FCYS medium
20 mMfructose and 30 mMsodium bicarbonate Cells were grown subsequently at 30C in full screw-capped bottles under anaerobic conditions in the dark
Preparation of membranes All steps were performed at 4C To obtain the membranes, cells (10 g wet-weight)were suspended in 80 mL of 30 mM Tris/HCl (pH 8.0), containing 20 mM sucrose and 1 mM phenylmethylsulfonyl fluoride Cells were treated with
5 mM 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 mM Tris/HCl (pH 8.0), the membrane component was centrifuged at 170 000 g for 60 min, and the pellet was suspended in 30 mM Tris/HCl (pH 8.0) containing 0.4M sucrose This membrane suspension was stored at)80 C until use
Purification of complex II All steps were performed at 4C To solubilize complex II, crude membranes (100 mg protein)were suspended at
1 mgÆmL)1protein in 10 mMTris/HCl (pH 7.5), 1% (w/v) polyoxyethylene-9-lauryl ether (C12E9)(Sigma)and 1 mM 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 mMTris/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.
Trang 3sucrose After washing with 40 mL of equilibration buffer
and 80 mL of equilibration buffer containing 50 mMNaCl,
complex II was eluted with 120 mL of equilibration buffer
containing a linear gradient of 50–150 mMNaCl 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 of membranes 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 mMTris/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 NaN3(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 NaBH4in methanol/0.1M
at room temperature produced crude n-decylrhodoquinol After oxidation of n-decylrhodoquinol to n-decylrhodo-quinone with Ag2O in ethyl acetate, the final product was purified by silica gel column chromatography (hexane/ CHCL3/AcOEt, 70 : 25 : 5, 82% yield) 1H NMR (CDCl3,
300 MHz) d 0.88 (t, J¼ 6.7 Hz, 3H, CH3), 1.2–1.5 (m, 16H, -CH2-) , 1.97 (s, 3H, ArCH3) , 2.45 (t, J ¼ 7.3 Hz, 2H, ArCH2-) , 3.87 (s, 3H, OCH3) , 4.67 (br s, 2H, NH2) ESIMS (m/z)308.3 [M + H]+
Measurement of enzymatic activities All assays were performed at 30C in 50 mM phosphate buffer (pH 7.5)containing 0.05% (w/v)sucrose mono-laurate SQR activity was measured as described previously [26] using 100 lM 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 · 6 cm)
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)1and 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 Elution was performed in the same
buffer at a flow rate of 3.3 mLÆh)1and 500 lL
fractions were collected.
Trang 4presence of PMS [27], using an extinction
coeffi-cient of 17 mM )1Æcm)1 for MTT To measure RQ-QFR
activity, synthetic n-decylrhodoquinone (100 lM)was used
as substrate [4] RQ-QFR activity was measured
anaero-bically in a rubber-stopped cuvette containing 10 mM
glucose, glucose oxidase (100 lgÆmL)1)(Boehringer
Mann-heim)and catalase (2 lgÆmL)1)(Wako)[28] and N2
-trea-ted buffer RQ was reduced by 1 mMsodium borohydride
before each assay, and the enzymatic reaction was initiated
by addition of 5 mMsodium fumarate Activity was
meas-ured by monitoring the change in absorbance of RQ at
283 nm, using an extinction coefficient of 7.9 mM )1Æcm)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-PSQ 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 encodingR 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 lM each)and genomic DNA were combined with
5 lL of 10· Taq polymerase buffer (Boehringer
Mann-heim), 200 lM of dNTP, and 2 lM of magnesium
chloride The volume was brought up to 50 lL with
distilled water, and 2.5 U of Taq DNA polymerase
(Boehringer Mannheim)were added Each amplification
cycle consisted of DNA denaturation at 95C for
0.5 min, primer annealing at 50C for 0.5 min, and
extension at 72C 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 byCLUSTAL 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 usingCLUSTAL 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 ofR 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)1Æmg)1 protein)was much higher than that in the mitochondria of the adult parasitic nematode A suum (26 nmolÆmin)1Æmg)1 protein) This finding indicates that RQ functions as an electron donor for fumarate reduction in R fermentans
Purification of complex II fromR 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 mM 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
Trang 5activity to 584 nmol min)1Æmg)1, and a 2.8-fold increase in
SQR specific activity to 4016 nmolÆmin)1Æmg)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 porobserva-tion of the enzyme complex dissociated into catalytic and membrane anchor domains during purification
Subunit composition and properties ofR 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
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)1protein),
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] (Table 2) 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 Km 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 coliSQR 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 complexII from anaerobically cultured R fermentans.
Total activity (nmolÆmin)1)
Specific activity (nmolÆmin)1Æmg)1)
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 complexII.
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).
Trang 6R 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 encodingR 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
in the same order as the E 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
complexII 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 complexII V max (lmolÆmin)1Æmg)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
V max
K m (l M )
V max
K m (l M )
QFR/SQR a
R fermentans complex II 1.17 35.7 39.9 7.30 262 6.2 0.160
a
V for RQ-QFR/V for SQR.bPurified from adult A suum mitochondria [60],cPurified from E coli GO103 [61].
Fig 5 Organisation of genes encoding R fermentans complexII 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.
Trang 7147, 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 complexII (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).
Trang 8N-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]
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 coliSQR [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-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 complex II shares a
com-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-1mutant 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 complexII.
Enzyme
Amino acid sequence identity
to R fermentans complex II (%)
References
Fp 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]
Trang 9biosynthesis 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, and A 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).
Trang 10This 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 relastructure-func-tionships 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 Rhodocyclus gelatinosus-like 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 reduc(1998)Characteriza-tion 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) Purification and properties of cytochrome b 556 in the respiratory chain of aero-bically grown Escherichia coli J Biol Chem 253, 8910–8915.