Novel Mitochondrial Complex II Isolated from Trypanosoma cruzi Is Composed of 12 Peptides Including a Heterodimeric Ip Subunit*

Mitochondrial respiratory enzymes play a central role in energy production in aerobic organisms. They differentiated from the α-proteobacteria-derived ancestors by adding noncatalytic subunits. An exception is Complex II (succinate: ubiquinone reductase), which is composed of four α-proteobacteria-derived catalytic subunits (SDH1-SDH4). Complex II often plays a pivotal role in adaptation of parasites in host organisms and would be a potential target for new drugs. We purified Complex II from the parasitic protist Trypanosoma cruzi and obtained the unexpected result that it consists of six hydrophilic (SDH1, SDH2N, SDH2C, and SDH5-SDH7) and six hydrophobic (SDH3, SDH4, and SDH8-SDH11) nucleus-encoded subunits. Orthologous genes for each subunit were identified in Trypanosoma brucei and Leishmania major. Notably, the iron-sulfur subunit was heterodimeric; SDH2N and SDH2C contain the plant-type ferredoxin domain in the N-terminal half and the bacterial ferredoxin domain in the C-terminal half, respectively. Catalytic subunits (SDH1, SDH2N plus SDH2C, SDH3, and SDH4) contain all key residues for binding of dicarboxylates and quinones, but the enzyme showed the lower affinity for both substrates and inhibitors than mammalian enzymes. In addition, the enzyme binds protoheme IX, but SDH3 lacks a ligand histidine. These unusual features are unique in the Trypanosomatida and make their Complex II a target for new chemotherapeutic agents.

Parasitic nematodes adapted to hypoxic host environments often have modified respiratory chains. Many adult parasites perform fumarate respiration by expressing a stage-specific isoform of Complex II (9,10). Hemonchus contortus uses an isoform for SDH2 (9), whereas Ascaris suum uses isoforms for SDH1 and SDH4 (10). To explore the adaptive strategy in a parasitic protist, we isolated mitochondria from axenic culture of T. cruzi epimastigotes and characterized the purified Complex II. Our results demonstrated for the first time that T. cruzi Complex II is an unusual supramolecular complex with a heterodimeric iron-sulfur subunit and seven novel noncatalytic subunits. Purified enzyme showed reduced binding affinities for both substrates and inhibitors. Because this novel structural organization is conserved in all trypanosomatids (2,15,16), parasite Complex II would be a potential target for the development of new chemotherapeutic agents for trypanosomiasis and leishmaniasis.

EXPERIMENTAL PROCEDURES
Preparation of Mitochondria-T. cruzi strain Tulahuhen was grown statically for 6 -7 days at 26°C in 300-cm 2 cell culture flasks (Falcon, BD Biosciences) containing 250 ml of the modified LIT medium (17), supplemented with 0.1% (w/v) glucose, 0.001% (w/v) hemin (Sigma), and 5% (v/v) fetal bovine serum (MP Biochemicals). Mitochondria were isolated from epimastigotes by the differential centrifugation method (18) with slight modifications. Parasites grown to 6 -8 ϫ 10 7 cells/ml were washed with buffer A (20 mM Tris-HCl, pH 7.2, 10 mM NaH 2 PO 4 , 1 mM sodium EDTA, 1 mM dithiothreitol, 0.225 M sucrose, 20 mM KCl, and 5 mM MgCl 2 ). Cells were disrupted by grinding with silicon carbide (Carborundum 440 mesh; Nacalai Tesque, Kyoto, Japan) in the presence of a minimum volume of buffer B (25 mM Tris-HCl, pH 7.6, 1 mM dithiothreitol, 1 mM sodium EDTA, 0.25 M sucrose, and EDTA-free Complete protease inhibitor mixture (Roche Applied Science)). The resultant cell paste was resuspended in buffer B and centrifuged at 500 ϫ g for 5 min and 1000 ϫ g for 15 min to remove silicon carbide and nuclear fraction, respectively. The mitochondrial fraction was recovered upon centrifugation of the last supernatant at 10,000 ϫ g for 15 min, washed three times in buffer B, and resuspended to a protein concentration of ϳ30 mg/ml and kept at Ϫ80°C until use.
Isolation of Complex II-All steps were carried out at 4°C. Mitochondrial fraction (ϳ300 mg of protein from 10 liters culture) was brought to 70 ml with buffer C (10 mM KP i , pH 7.5), 1 mM sodium EDTA, 1 mM sodium malonate, EDTA-free Complete protease inhibitor mixture (Roche Applied Science) (2 tablets/50 ml), 1% (w/v) sucrose monolaurate SM-1200 (SML) (Mitsubishi-Kagaku Foods Co., Tokyo, Japan)). The mixture was stirred for 30 min and centrifuged at 200,000 ϫ g for 1 h. The supernatant was loaded at 1 ml/min onto a Source 15 Q column (1.6 inner diameter ϫ 10 cm; GE Healthcare), equilibrated with buffer C containing 0.1% SML. After washing with 5 volumes of the same buffer, proteins were eluted with a 200-ml linear gradient of NaCl from 0 to 150 mM at 2 ml/min. Active fractions were concentrated to ϳ250 l by ultrafiltration with Amicon Ultra-4 (molecular weight cutoff 100,000, Millipore) and subjected to gel filtration FPLC with a Superdex 200-pg 10/300 GL column (1 cm inner diameter ϫ 30 cm; GE Healthcare) at 0.25 ml/min in 20 mM MOPS-NaOH, pH 7.2, containing 1 mM sodium EDTA, 1 mM sodium malonate, 150 mM NaCl, and 0.1% SML. Peak fractions were rechromatographed as above, and purified enzyme was concentrated and stored at Ϫ80°C until use.
Identification of Complex II Subunits-The purified enzyme was subjected to 12.5% SDS-PAGE, and subunits were transferred to an Immobilon-P membrane (Millipore), followed by FIGURE 1. Metabolic pathways in T. cruzi. Incomplete oxidation of glucose takes place in glycosomes and mitochondria, and end products such as succinate, L-alanine, ethanol, and acetate are excreted from parasites (3,4). Cytoplasmic dihydroorotate (DHO):fumarate reductase (DHOD) contributes succinate production (6). Complex II fractions from the first gel filtration chromatography with a Superdex 200-pg column were concentrated and rechromatographed at the flow rate of 0.25 ml/min. Aliquots were collected every 0.5 ml. Elution profiles for proteins and cytochromes were monitored at 280 (OE) and 412 nm (E), respectively, and the enzyme activity (F) was measured as decylquinonemediated succinate:DCIP reductase.  (20). Genes coded for Complex II subunits were identified with BLASTP in the T. cruzi genome data base (15). Phase Partitioning of Mitochondrial Fraction with Triton X-114-Phase partitioning by Triton X-114 was performed as described previously (21) with a slight modification. A total of 2-3 mg of mitochondrial fraction was resuspended in 1 ml of Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 2 mM sodium malonate, Complete protease inhibitors mixture (Roche Applied Science) (2 tablets/50 ml), protease inhibitors mixture for mammalian cell and tissue extracts (Sigma) (10 l/ml), and 2% (v/v) Triton X-114. The mixture was incubated for 30 min on ice and kept at Ϫ30°C overnight. After thawing, the insoluble material was removed by centrifugation at 4°C, and the supernatant was incubated for 10 min at 37°C and centrifuged at 2000 ϫ g for 10 min to separate the aqueous and detergentrich phases. The aqueous phase was brought to 2% (v/v) Triton X-114, whereas the detergent-rich fraction was brought to 1 ml with the above buffer. After incubation on ice for 10 min, samples were incubated at 37°C for 10 min and phases separated as before. This wash step was repeated three times. Finally, the samples were dialyzed and concentrated by Amicon Ultra-4 (Millipore) in the presence of 50 mM imidazole, 50 mM NaCl, 6 mM aminocaproic acid, 0.05% (w/v) deoxycholate, and 0.1% (w/v) SML, pH 7, and kept at Ϫ80°C until use.
Miscellaneous-High resolution clear native electrophoresis (hrCNE) (22) was performed with 4 -16% Novex gels (Invitrogen) using 0.02% dodecylmaltoside and 0.05% sodium deoxycholate for the cathode buffer additives, and the Complex II band was visualized by the activity staining (23) or Coomassie Brilliant Blue. Tricine-PAGE analysis was done with Novex 10 -20% Tricine gels (Invitrogen), and proteins bands were sequentially stained by Sypro ruby (Invitrogen) and silver. During purification the succinate-decylubiquinone-DCIP reduc-  a Alleles were named as SDH3-1 (XP_809410) and SDH3-2 (XP_810064) in the order of the accession numbers, except for SDH5. b These are N-terminal sequences except for SDH2 N and SDH8, where the N-terminal residues were blocked. c Homozygous alleles located in a merged assembly of Esmeraldo (E) and non-Esmeraldo (NE) homologous sequences whose different copies were merged genes during the genome assembly are indicated by "merged." Haplotypes for gene with more than two copies in the genome that does not belong to a merged region are not assigned (NA). d Identity % to counterparts in human were as follows: SDH1 (D30648), SDH2 (P21912), SDH3 (Q99643), or SDH4 (O14521). In parentheses, the identity % of SDH2 N and SDH2 C that correspond to either Met 1 -Pro 155 (Ip N domain) or Tyr 156 -Val 280 (Ip C domain), respectively, of human SDH2 is shown. Identity % for truncated forms of SDH1 and SDH5 (SDH1-2 and SDH5-2) in the Esmeraldo haplotype was 66 and 20%, respectively. e Transmembrane segments (TM) were estimated with TMHMM (52) and SOSUI (53). f SDH2N from other trypanosomatids lack Met 1 to Arg 42 of TcSDH2 N . g ND indicates not determined because these hydrophobic sequences are a highly divergent form of mammalian sequences. MARCH 13, 2009 • VOLUME 284 • NUMBER 11 tase activity was monitored in a microplate spectrophotometer (Benchmark Plus, Bio-Rad). Kinetics and UV-visible absorption spectra were determined at room temperature with a V-660 UV-visible spectrophotometer (Jasco, Tokyo, Japan). Protoheme IX and protein concentrations were determined by pyridine hemochromogen method (24) and the micro BCA method (Pierce), respectively. Sequence alignment was done with ClustalX 2.0 (25).

RESULTS AND DISCUSSION
Isolation of T. cruzi Complex II-To determine the molecular organization of T. cruzi Complex II, we purified this enzyme from epimastigote mitochondria by ion-exchange and gel filtration chromatography using the nonionic detergent sucrose monolaurate (Table 1). Decylubiquinone-mediated succinate: DCIP reductase activity was eluted as a single peak at each step

12-Subunit Complex II from T. cruzi
and co-eluted with proteins and b-type cytochrome(s) at the second Superdex 200 chromatography (Fig. 2). Specific activity was increased 34-fold to 2.9 units/mg proteins, and the yield was ϳ2%. A hrCNE of the pure protein identified T. cruzi Complex II as an ϳ550-kDa complex (Fig. 3, lanes 1 and 3), which is 4-fold larger than bovine and yeast Complex II (130 kDa) and potato Complex II (150 kDa) (26,27). Upon phase partitioning of the mitochondrial fraction with Triton X-114, the Complex II of T. cruzi was found only in the detergent-rich fraction (data not shown). Analysis of the detergent-rich fraction by hrCNE showed the Complex II as a single band at the same position as the pure enzyme (ϳ550 kDa) (Fig. 3, lanes 2 and 4). These results indicated that the purified Complex II was obtained in its intact form. Interestingly, second dimensional analysis of both the purified Complex II and the detergent-rich fraction from phase partitioning with Triton X-114 with SDH activity showed that T. cruzi Complex II is composed of 12 subunits (Fig. 3, lanes 5 and 6). The same subunit composition was obtained by immunoaffinity purification of the partially purified enzyme (data not shown). The apparent molecular weight of the subunits ranges from 7.3 to 63 kDa (Fig. 3, lanes 7 and 8).
Assuming the presence of equimolar amounts of subunits, a total molecular mass of Complex II would be 286.5 kDa, indicating that T. cruzi Complex II is a homodimer.
Iron-Sulfur Subunit-Sequence analysis of the 25-and 21-kDa band proteins revealed that they contain the plant ferredoxin domain (Ip N ) and bacterial ferredoxin domain (Ip C ) of canonical SDH2 (Ip) in the N-and C-terminal half, respectively (Fig. 4). Sequence identities of Ip N and Ip C are 37 and 43%, respectively, to those of human SDH2 (Table 2), and the Ip N and Ip C domains contain all amino acid residues responsible for binding of iron-sulfur clusters and ubiquinone (12,13,30) (Fig. 4). Such a heterodimeric Ip subunit can be found in T. brucei (31), T. cruzi, L. major, L. infantum, and L. brasiliensis (Tables 2), which belong to the order Trypanosomatida. Thus we named these subunits as SDH2 N and SDH2 C , respectively.
Splitting of mitochondrial membrane proteins has been reported for cytochrome c oxidase CoxII in Apicomplexa and Chlorophyceae (32,33), and ATP synthase ␣ subunit in Leishmania tarentolae and T. brucei (34,35). The former occurs at the gene level and the latter by post-translational cleavage. Sequence analysis indicates that heterodimeric SDH2 and CoxII have emerged from gene duplication followed by degeneration of the N-or C-terminal half of the duplication products. Conserved domains in degenerated duplicons, which have arisen from mitochondrion-to-nucleus transfer of the dupli-cated genes (32,33,36), must retain the potential for proteinprotein interactions and constitute a heterodimeric functional subunit by trans-complementation.
Membrane Anchor Subunits-Membrane anchor subunits in protist enzymes are highly divergent from bacterial and mammalian counterparts and difficult to find with conventional BLAST programs. We identified candidates for T. cruzi SDH3 and SDH4 by the presence of the quinone/heme-binding motifs "RPX 16 SX 2 HR (SDH3 helix I)" and "HX 10 DY (SDH4 helix V)," respectively, present in membrane anchor subunits. In Complex II, Trp 164 in SDH2 (Fig. 4) and Tyr 83 in the SDH4 HX 10 DY motif (Fig. 5B) (E. coli numbering) could hydrogen bond to the O-1 atom of ubiquinone and contribute to the binding affinity (12,37). Arg 31 in the SDH3 SX 2 HR motif (Fig. 5A) and Asp 82 in the SDH4 HX 10 DY motif are in close proximity to ubiquinone and could interact with Tyr 83 (37). Ser 27 in the SDH3 SX 2 HR motif has been shown to be essential for quinone binding (38) and is a candidate for hydrogen bonding to the O-4 atom of ubiquinone (30). The first arginine (Arg 9 in E. coli SDH3) in the RPX 16 SX 3 R motif is in the vicinity of Glu 186 in SDH1 and Asp 106 in SDH2 and may play a structural role by making a hydrogen bond network.
In T. cruzi, SDH3 has the "RPX 11 SX 2 HR motif in front of the predicted transmembrane helix I and lacks transmembrane helices II and III. However, sequence alignment suggests the presence of the alternative motif "TX 2 SR/(T)" in the Trypanosomatida (Fig. 5A). In mitochondrial Complex II, protoheme IX is ligated by two His residues in the second transmembrane helix of SDH3 ("HX 10 D" motif) and SDH4 ("HX 10 DY" motif). A heme ligand in helix II (His 84 in E. coli SDH3) may be substituted by a nearby histidine in the quinone-binding motif "SX 2 HR" (39). In contrast, SDH4 lacks helix IV and appears to interact with heme and ubiquinone with the HX 10 DY motif. As in rice SDH4 (GenBank TM accession number NP_001045324), the heme ligand His is substituted by Gln in T. brucei SDH4. The presence of a bound heme or an alternative ligand in T.

12-Subunit Complex II from T. cruzi
brucei SDH4 needs to be tested in future studies. It is also possible that trypanosomatid-specific subunits could be assembled as a jigsaw puzzle-like membrane anchor.
Spectroscopic Properties of T. cruzi Complex II-Pyridine ferrohemochrome analysis showed that T. cruzi Complex II binds a stoichiometric amount of protoheme IX (0.85 heme/monomer of enzyme) indicating that monomer enzyme complex contains one heme. At room temperature, the air-oxidized and fully reduced forms of the purified enzyme showed peaks at 413 and 426, 527, and 561 nm, respectively (Fig. 6). Peak positions are similar to those reported for Complex II from E. coli (40), adult A. suum (41), and bovine (42,43), where heme is ligated via histidine in the second helices of SDH3 and SDH4. Although heme has an important role in the assembly of Complex II, it is not essential for the reduction of ubiquinones (43,44).
Enzymatic Properties of T. cruzi Complex II-We examined SQR activity of the purified enzyme and found the difference in apparent K m values between Q 1 (33.9 Ϯ 3.6 M) and Q 2 (18.8 Ϯ 6.4 M) (Fig. 7), indicating that the 6-polyprenyl group of ubiquinone contributes to the binding affinity. The apparent V max value of the T. cruzi Complex II was rather constant, 11.9 Ϯ 2.2 for Q 1 and 11.5 Ϯ 0.4 Q 2 units/mg proteins, respectively, and one-fourth of those reported for bovine and E. coli enzymes (45,46). This is not surprising because T. cruzi complex II has about 2-3 times more proteins than the other enzymes. K m values for ubiquinone and succinate (18.8 Ϯ 6.4 M (Q 2 ) and 1.48 Ϯ 0.17 mM, respectively) were higher than 0.3 and 130 M, respectively, of bovine enzyme (45), and 2 and 277 M, respectively, of the E. coli enzyme (46,47). Notably, the K m value for succinate was comparable with 610 M in adult A. suum (10), which expresses the stage-specific Complex II as quinol:fumarate reductase under hypoxic habitats in host organisms.
Then we examined effects of inhibitors for binding sites of quinones and dicarboxylates on SQR activity. Atpenin A5, a potent inhibitor for Complex II, inhibited the T. cruzi enzyme with the IC 50 value of 6.4 Ϯ 2.4 M, which is 3 orders of magnitude higher than that of bovine Complex II (4 nM) (48). Furthermore, carboxin, 2-theonyltrifluoroacetone, plumbagin, and 2-heptyl-4-hydroxyquinoline N-oxide were ineffective (100 M ϽIC 50 ). Structural divergence in trypanosomatid SDH3 and SDH4 could be the cause for lower binding affinities for both quinones and inhibitors. In addition, we found for the dicarboxylate-binding site that the IC 50 value for malonate (40 M) was much higher than the K i value for bovine Complex II (1.3 M) (45).
Structure of Trypanosomatid Complex II-To the best of our knowledge, this is the first report on the isolation of protist Complex II. T. cruzi Complex II has unusual subunit organization with six each of hydrophilic and hydrophobic subunits. Such a supramolecular structure and heterodimeric SDH2 (SDH2 N and SDH2 C ) are conserved in the Trypanosomatida. Furthermore, SDH1, SDH2 N , SDH2 C , SDH3, SDH4, and SDH8 -SDH10 can be identified in the ongoing genome projects on the evolutionary relatives, the photosynthetic freeliving Euglena gracilis, and the nonphotosynthetic euglenoid Astasia longa in the Euglenida. Thus a part of these features are common in the Euglenozoa, a divergent lineage of eukaryotes (Fig. 8).
Accumulation of noncatalytic subunits through expanding the protein interaction network could be a driving force for protein evolution. Structural and catalytic features are unique, and thus this enzyme could be a potential target for novel chemotherapeutic agents for trypanosomiasis and leishmaniasis.
Conclusion-The parasitic protist T. brucei is a gold mine where unprecedented biological phenomena like RNA editing and transsplicing in mitochondria were originally discovered. It was found recently in Diplonema papillatum, a free-living evolutionary cousin,   MARCH 13, 2009 • VOLUME 284 • NUMBER 11 that mature mRNA for cytochrome c oxidase CoxI was assembled from nine gene fragments by a jigsaw puzzle mechanism (49). From a characterization of Complex II from T. cruzi, we revealed a novel supramolecular organization, which is conserved in the Trypanosomatida.

12-Subunit Complex II from T. cruzi
Parasites have exploited unique energy metabolic pathways as adaptations to their natural habitats within their hosts (50,51). In fact, the respiratory systems of parasites typically show greater diversity in electron transfer pathways than those of host animals. As shown in this study, such is also the case with Complex II, which is a well known marker enzyme of mitochondria. Studies on the role of supramolecular Complex II in adaptation of trypanosomatids is now underway in our laboratory.