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

Abstract

Mitochondrial respiratory enzymes play a central role in energy production in aerobic organisms. They differentiated from the ␣-proteobacteria-derived ancestors by adding noncatalytic subunits. An exception is Complex II (succinate: ubiquinone reductase), which is composed of four ␣-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, SDH2 N , SDH2 C , 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; SDH2 N and SDH2 C 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, SDH2 N plus SDH2 C , 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.The parasitic protist Trypanosoma cruzi is the etiological agent of Chagas disease, a public health threat in Central and South America. These parasites are normally transmitted by reduviid bugs via the vector feces after a bug bite and also via transfusion of infected blood. About 16 -18 million people are infected, and 100 million are at risk, but there are no definitive chemotherapeutic treatments available (1). Despite having potential pathways for oxidative phosphorylation (2), all trypanosomatids (Trypanosoma and Leishmania species) analyzed so far are characterized by incomplete oxidation of glucose with secretion of end products, such as succinate, alanine, ethanol, acetate, pyruvate, and glycerol (3, 4) (Fig. 1). Major routes for formation of succinate in Trypanosoma brucei are via NADH-dependent fumarate reductase in glycosomes and mitochondria (5, 6). In trypanosomatid mitochondria, the Krebs cycle is inefficient, and pyruvate is principally converted to acetate via acetate:succinate CoA transferase (7). A part of the Krebs cycle operates the utilization of histidine in the insect stage of T. cruzi (8).Mitochondrial Complex II (succinate:quinone reductase (SQR) 5 and succinate dehydrogenase (SDH)) serves as a membrane-bound Krebs cycle enzyme and often plays a pivotal role in adaptation of parasites to environments in their host (9, 10). In general, Complex II consists of four subunits (11). A flavoprotein subunit (SDH1, Fp) and an iron-sulfur subunit (SDH2, Ip) form a soluble heterodimer, which then binds to a membrane anchor heterodimer, SDH3 (CybL) and SDH4 (CybS)....

The mitochondrial respiratory chain of the secondary green alga Euglena gracilis shares many additional subunits with parasitic Trypanosomatidae

“…This further illustrates the difficulty to assess orthology for small hydrophobic membrane proteins [see also Discussion in (Cardol, 2011)]. Additional subunits were also found in trypanosomes (Acestor et al, 2011;Morales et al, 2009), some of them also present in E. gracilis.…”

“…brucei SDH3 and SDH4 were proposed based on the presence of quinone/heme-binding motifs, predicted trans-membrane domains and similar protein sizes, but a direct comparison with these proteins did not reveal any obvious predicted structural or motif similarity (Acestor et al, 2011;Morales et al, 2009). This further illustrates the difficulty to assess orthology for small hydrophobic membrane proteins [see also Discussion in (Cardol, 2011)].…”

“…Compared with their prokaryotic counterparts, mitochondrial respiratory-chain complexes have acquired additional subunits, although most of these are not directly involved in the catalytic activity of the enzyme complexes. During the last decade, many studies have highlighted the peculiarities of respiratory-chain complexes in trypanosomes , Morales, Mogi et al 2009, Zikova, Schnaufer et al 2009, Acestor, Zikova et al 2011, in which the majority of the identified subunits are apparently unrelated to those described in green plants, mammals or fungi.…”

“…CII usually comprises four subunits (SDH1-4): a flavoprotein (SDH1), an iron-sulfur (Fe-S) protein (SDH2) and two membrane subunits (SDH3/SDH4) providing ligands to heme b and a reduction site for ubiquinone. As previously pointed out (Morales et al, 2009), membrane anchor subunits are highly divergent between bacteria, mammals and other eukaryotes, and thus are difficult to identify with standard BLAST searches (Acestor et al, 2011). Candidates for T. cruzi and T.…”

“…On the one hand, an alternative electron transfer chain, composed of a glyceraldehyde-3-phosphate dehydrogenase (G3PD) and an alternative oxidase (TAO or AOX), replaces the classical complexes I-IV during the bloodstream (or mammalianinfective) stage (Chaudhuri et al, 2006). On the other hand, respiratory complexes I-IV and the ATP synthase (complex V) found in the procyclic (or insect-infective) stage of trypanosomes possess many apparently-specific additional subunits (Acestor et al, 2011;Morales et al, 2009;Panigrahi et al, 2008;Zikova et al, 2008;Zikova et al, 2009). …”