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: 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-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. 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Complex II from phototrophic purple bacterium Rhodoferax fermentans displays rhodoquinol-fumarate reductase activity Hiroko Miyadera 1, *,. purple bacterium, Rhodoferax fermentans that exhibited high rhodoquinol-fumarate reductase activity in addition to succinate-ubiquinone reductase activity.