Toward understanding the role of mitochondrial complex II in the intraerythrocytic stages of Plasmodium falciparum: Gene targeting of the Fp subunit

Malaria parasites in human hosts depend on glycolysis for most of their energy production, and the mitochondrion of the intraerythrocytic form is acristate. Although the genes for all tricarboxylic acid (TCA) cycle members are found in the parasite genome, the presence of a functional TCA cycle in the intraerythrocytic stage is still controversial. To elucidate the physiological role of Plasmodium falciparum mitochondrial complex II (succinate-ubiquinone reductase (SQR) or succinate dehydrogenase (SDH)) in the TCA cycle, the gene for the flavoprotein subunit (Fp) of the enzyme, pfsdha (P.falciparum gene for SDH subunit A, PlasmoDB ID: PF3D7_1034400) was disrupted. SDH is a well-known marker enzyme for mitochondria. In the pfsdha disruptants, Fp mRNA and polypeptides were decreased, and neither SQR nor SDH activity of complex II was detected. The suppression of complex II caused growth retardation of the intraerythrocytic forms, suggesting that complex II contributes to intraerythrocytic parasite growth, although it is not essential for survival. The growth retardation in the pfsdha disruptant was rescued by the addition of succinate, but not by fumarate. This indicates that complex II functions as a quinol-fumarate reductase (QFR) to form succinate from fumarate in the intraerythrocytic parasite.     

Direct evidence for expression of type II flavoprotein subunit in human complex II (succinate-ubiquinone reductase)

Succinate-ubiquinone reductase (complex II) is an important enzyme complex in aerobic respiration and the tricarboxylic acid cycle. We recently identified two distinct cDNAs for the human flavoprotein subunit (Fp) from a single individual and demonstrated mRNAs of these two isoforms, Type I Fp and Type II Fp, in skeletal muscle, liver, brain, heart, and kidney. Type I Fp was expressed at higher levels than Type II Fp in all cases. In the present study, the biochemical properties of Type II Fp-containing complex II in Raji cells predominantly expressing Type II Fp were investigated. Complex II having Type II Fp was separated from that having Type I Fp by isoelectric focusing in the presence of sucrose monolaurate. Together with the fact that succinate-ubiquinone reductase activity of mitochondria prepared from Raji cell was almost identical to that from human liver, these results clearly indicate the presence of two distinct isoforms of active complex II in human mitochondria.     

Crystal structure of mitochondrial quinol-fumarate reductase from the parasitic nematode Ascaris suum

In the anaerobic respiratory chain of the parasitic nematode Ascaris suum, complex II couples the reduction of fumarate to the oxidation of rhodoquinol, a reverse reaction catalyzed by mammalian complex II. In this study, the first structure of anaerobic complex II of mitochondria was determined. The structure, composed of four subunits and five co-factors, is similar to that of aerobic complex II, except for an extra peptide found in the smallest anchor subunit of the A. suum enzyme. We discuss herein the structure-function relationship of the enzyme and the critical role of the low redox potential of rhodoquinol in the fumarate reduction of A. suum complex II.     

Complex II from phototrophic purple bacterium Rhodoferax fermentans displays rhodoquinol-fumarate reductase activity.

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 benzoquinone, 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 reductase. The results strongly indicate that R. fermentans complex II and mitochondrial QFR might have evolved independently, although they both utilize RQ for fumarate reduction.     

Crystal structure of mitochondrial respiratory membrane protein complex II

The mitochondrial respiratory Complex II or succinate:ubiquinone oxidoreductase (SQR) is an integral membrane protein complex in both the tricarboxylic acid cycle and aerobic respiration. Here we report the first crystal structure of Complex II from porcine heart at 2.4 A resolution and its complex structure with inhibitors 3-nitropropionate and 2-thenoyltrifluoroacetone (TTFA) at 3.5 A resolution. Complex II is comprised of two hydrophilic proteins, flavoprotein (Fp) and iron-sulfur protein (Ip), and two transmembrane proteins (CybL and CybS), as well as prosthetic groups required for electron transfer from succinate to ubiquinone. The structure correlates the protein environments around prosthetic groups with their unique midpoint redox potentials. Two ubiquinone binding sites are discussed and elucidated by TTFA binding. The Complex II structure provides a bona fide model for study of the mitochondrial respiratory system and human mitochondrial diseases related to mutations in this complex.     

Free-living nematodes Caenorhabditis elegans possess in their mitochondria an additional rhodoquinone, an essential component of the eukaryotic fumarate reductase system.

The respiratory chain of Caenorhabditis elegans was characterized in mitochondria isolated from aerobically grown nematodes. Nematode mitochondria contain ubiquinone-9 as a major component and rhodoquinone-9 as a minor component. The ratio of ubiquinone-9/rhodoquinone-9 is higher in C. elegans mitochondria than in mitochondria from second-stage larvae of Ascaris suum, the free-living stage of porcine gut-dwelling nematode. The individual oxidoreductase activities comprising succinate oxidase and the amount of substrate-reducible cytochromes are comparable to those of mitochondria from second-stage larvae of A. suum. The specific activity of fumarate reductase is lower in C. elegans mitochondria than in mitochondria from second-stage larvae of A. suum, but still higher than in mammalian mitochondria. These results indicate that the free-living nematode C. elegans is capable of synthesizing rhodoquinone, as distinguished from aerobic mammalian species, although its mitochondria appear more aerobic than A. suum larval mitochondria.     

Direct evidence for two distinct forms of the flavoprotein subunit of human mitochondrial complex II (succinate-ubiquinone reductase)

Succinate-ubiquinone reductase (complex II) is an important enzyme complex in both the tricarboxylic acid cycle and aerobic respiration. A recent study showed that defects in human complex II are associated with cancers as well as mitochondrial diseases. Mutations in the four subunits of human complex II are associated with a wide spectrum of clinical presentations. Such tissue-specific clinical symptoms suggest the presence of multiple isoforms of the subunits, but subunit isoforms have not been previously reported. In the present study, we identified two distinct cDNAs for the human flavoprotein subunit (Fp) from a single individual, and demonstrated expression of these two isoforms in skeletal muscle, liver, brain, heart and kidney. Interestingly, one of the Fp isoforms was encoded as an intronless gene.     

A third crystal form of Wolinella succinogenes quinol:fumarate reductase reveals domain closure at the site of fumarate reduction.

Quinol:fumarate reductase (QFR) is a membrane protein complex that couples the reduction of fumarate to succinate to the oxidation of quinol to quinone. Previously, the crystal structure of QFR from Wolinella succinogenes was determined based on two different crystal forms, and the site of fumarate binding in the flavoprotein subunit A of the enzyme was located between the FAD-binding domain and the capping domain [Lancaster, C.R.D., Kr?ger, A., Auer, M., & Michel, H. (1999) Nature 402, 377--385]. Here we describe the structure of W. succinogenes QFR based on a third crystal form and refined at 3.1 A resolution. Compared with the previous crystal forms, the capping domain is rotated in this structure by approximately 14 degrees relative to the FAD-binding domain. As a consequence, the topology of the dicarboxylate binding site is much more similar to those of membrane-bound and soluble fumarate reductase enzymes from other organisms than to that found in the previous crystal forms of W. succinogenes QFR. This and the effects of the replacement of Arg A301 by Glu or Lys by site-directed mutagenesis strongly support a common mechanism for fumarate reduction in this superfamily of enzymes.     

Isolation of the cytochrome-bc1 complex from rat-liver mitochondria

The cytochrome bc1 complex has been isolated from rat-liver mitochondria by two different procedures. The enzyme isolated by either procedure exhibits a specific cytochrome b and cytochrome c1 heme content of approximately 8 and 4 nmol/mg protein respectively. Both preparations contain only seven polypeptides on sodium dodecylsulfate gel electrophoresis, with the following apparent molecular weights: I, 50000; II, 46000; III, 33000; IV, 25000; V, 12500; VI, 10000; VII, 5600. The polypeptide composition is identical to that of the beef-heart enzyme isolated by cholate/ammonium sulfate fractionation. Furthermore, with the exception of subunit II (core protein 2), the apparent molecular weights of the subunits are identical in the rat-liver and beef-heart enzymes.     

Fumarate reductase and succinate oxidase activity of Escherichia coli complex II homologs are perturbed differently by mutation of the flavin binding domain

The Escherichia coli complex II homologues succinate:ubiquinone oxidoreductase (SQR, SdhCDAB) and menaquinol:fumarate oxidoreductase (QFR, FrdABCD) have remarkable structural homology at their dicarboxylate binding sites. Although both SQR and QFR can catalyze the interconversion of fumarate and succinate, QFR is a much better fumarate reductase, and SQR is a better succinate oxidase. An exception to the conservation of amino acids near the dicarboxylate binding sites of the two enzymes is that there is a Glu (FrdA Glu-49) near the covalently bound FAD cofactor in most QFRs, which is replaced with a Gln (SdhA Gln-50) in SQRs. The role of the amino acid side chain in enzymes with Glu/Gln/Ala substitutions at FrdA Glu-49 and SdhA Gln-50 has been investigated in this study. The data demonstrate that the mutant enzymes with Ala substitutions in either QFR or SQR remain functionally similar to their wild type counterparts. There were, however, dramatic changes in the catalytic properties when Glu and Gln were exchanged for each other in QFR and SQR. The data show that QFR and SQR enzymes are more efficient succinate oxidases when Gln is in the target position and a better fumarate reductase when Glu is present. Overall, structural and catalytic analyses of the FrdA E49Q and SdhA Q50E mutants suggest that coulombic effects and the electronic state of the FAD are critical in dictating the preferred directionality of the succinate/fumarate interconversions catalyzed by the complex II superfamily.     

Purification of NADPH:cytochrome c (cytochrome P-450) reductase from hamster liver microsomes by detergent extraction and affinity chromatography

NADPH:cytochrome c (cytochrome P-450) reductase (Fp) from hamster liver microsomes has been purified to near homogeneity using a simple and rapid method. Microsomes were treated with the detergent Chaps (3-[(3-cholamidopropyl)dimethylammonio]propanesulfonic acid) in combination with 0.07% protamine sulfate and then centrifuged to pellet insoluble material. While over 60% of the total microsomal protein was solubilized, all Fp activity remained in the pellet. Fp was extracted from the Chaps-insoluble material using a combination of the detergents sodium cholate and Lubrol PX. This treatment resulted in a fivefold increase in Fp specific activity and allowed direct processing of the enriched Fp fraction by 2',5'-ADP agarose affinity chromatography. The purified Fp had a total flavin content of 23 nmol/mg protein (flavin adenine dinucleotide:flavin mononucleotide ratio = 1:1), a specific activity of 26,000 units/mg protein at 22 degrees C using cytochrome c as electron acceptor, and migrated as a single band on sodium-dodecyl sulfate-polyacrylamide gel electrophoresis with a relative molecular weight of 76,000. The purity, specific activity, and yield were nearly identical to results obtained when the flavoprotein was purified by conventional methods. This procedure eliminates the need for anion-exchange chromatography and allows for the rapid purification of large amounts of Fp suitable for use in studies concerning cytochrome P-450-mediated drug metabolism. Importantly, this method is equally effective when used to purify Fp from rat liver microsomes.     

Type II Fp of human mitochondrial respiratory complex II and its role in adaptation to hypoxia and nutrition-deprived conditions

The flavoprotein (Fp) subunit of human mitochondrial succinate-ubiquinone reductase (SQR, complex II) has isoforms (type I, type II). Type II Fp is predominantly expressed in some cancer and fetal tissues and those tissues are often exposed to ischemia. The present study shows that complex II with type II Fp has lower optimal pH than complex II with type I Fp, and type II Fp mRNA expression was induced by ischemia. The result suggests complex II with type II Fp may function in cells with low mitochondrial matrix pH caused by ischemia and its function is related to cellular adaptation to ischemia.     

Electron-transfer complexes of Ascaris suum muscle mitochondria. II. Succinate-coenzyme Q reductase (complex II) associated with substrate-reducible cytochrome b-558

A succinate-coenzyme Q reductase (complex II) was isolated in highly purified form from Ascaris muscle mitochondria by detergent solubilization, ammonium sulfate fractionation and gel filtration on a Sephadex G-200 column. The enzyme preparation catalyzes electron transfer from succinate to coenzyme Q1 with a specific activity of 1.2 mumol coenzyme Q1 reduced per min per mg protein at 25 degrees C. The isolated complex II is essentially free of NADH-ferricyanide reductase, reduced CoQ2-cytochrome c reductase and cytochrome c oxidase and consists of four major polypeptides with apparent molecular weights of 66 000, 27 000, 12 000 and 11 000 and two minor ones with Mr of 36 000 and 16 000. The complex II contained cytochrome b-558, a major constituent cytochrome of Ascaris mitochondria, at a concentration of 3.6 nmol per mg protein, but neither other cytochromes nor quinone. The cytochrome b-558 in the complex II was reduced with succinate. In the presence of Ascaris NADH-cytochrome c reductase (complex I-III) (Takamiya, S., Furushima, R. and Oya, H. (1984) Mol. Biochem. Parasitol. 13, 121-134), the cytochrome b-558 in complex II was also reduced with NADH and reoxidized with fumarate. These results suggest the cytochrome b-558 to function as an electron carrier between NADH dehydrogenase and succinate dehydrogenase in the Ascaris NADH-fumarate reductase system.     

Geometric restraint drives on- and off-pathway catalysis by the Escherichia coli menaquinol:fumarate reductase

Complex II superfamily members catalyze the kinetically difficult interconversion of succinate and fumarate. Due to the relative simplicity of complex II substrates and their similarity to other biologically abundant small molecules, substrate specificity presents a challenge in this system. In order to identify determinants for on-pathway catalysis, off-pathway catalysis, and enzyme inhibition, crystal structures of Escherichia coli menaquinol:fumarate reductase (QFR), a complex II superfamily member, were determined bound to the substrate, fumarate, and the inhibitors oxaloacetate, glutarate, and 3-nitropropionate. Optical difference spectroscopy and computational modeling support a model where QFR twists the dicarboxylate, activating it for catalysis. Orientation of the C2-C3 double bond of activated fumarate parallel to the C(4a)-N5 bond of FAD allows orbital overlap between the substrate and the cofactor, priming the substrate for nucleophilic attack. Off-pathway catalysis, such as the conversion of malate to oxaloacetate or the activation of the toxin 3-nitropropionate may occur when inhibitors bind with a similarly activated bond in the same position. Conversely, inhibitors that do not orient an activatable bond in this manner, such as glutarate and citrate, are excluded from catalysis and act as inhibitors of substrate binding. These results support a model where electronic interactions via geometric constraint and orbital steering underlie catalysis by QFR.     

Catalase activity during the development of the parasitic nematode, Ascaris suum

1. Catalase activity was partially purified from body wall muscle of the parasitic nematode, Ascaris suum, and was similar to catalases isolated from mammalian tissues. It exhibited a broad pH optimum and was unaffected by 2 mM ethylenediaminetetra-acetate. In contrast, it was inhibited reversibly by 1 mM cyanide and irreversibly by prior incubation in 40 mM 3-amino-1:2:4-triazole for 1 hr or heating at 80 degrees C for 15 min. 2. Catalase activity was highest in the unembryonated "egg" and decreased dramatically as development proceeded. 3. Catalase activity in adult body wall muscle was similar to that in rat skeletal muscle, but dramatically lower than that in rat liver. Catalase activity was barely detectable in A. suum testis. 4. Cytochrome-c peroxidase activity did not appear to be present in adult A. suum muscle mitochondria.     

Characterization of superoxide-producing sites in isolated brain mitochondria

Mitochondrial respiratory chain complexes I and III have been shown to produce superoxide but the exact contribution and localization of individual sites have remained unclear. We approached this question investigating the effects of oxygen, substrates, inhibitors, and of the NAD+/NADH redox couple on H2O2 and superoxide production of isolated mitochondria from rat and human brain. Although rat brain mitochondria in the presence of glutamate+malate alone do generate only small amounts of H2O2 (0.04 +/- 0.02 nmol H2O2/min/mg), a substantial production is observed after the addition of the complex I inhibitor rotenone (0.68 +/- 0.25 nmol H2O2/min/mg) or in the presence of the respiratory substrate succinate alone (0.80 +/- 0.27 nmol H2O2/min/mg). The maximal rate of H2O2 generation by respiratory chain complex III observed in the presence of antimycin A was considerably lower (0.14 +/- 0.07 nmol H2O2/min/mg). Similar observations were made for mitochondria isolated from human parahippocampal gyrus. This is an indication that most of the superoxide radicals are produced at complex I and that high rates of production of reactive oxygen species are features of respiratory chain-inhibited mitochondria and of reversed electron flow, respectively. We determined the redox potential of the superoxide production site at complex I to be equal to -295 mV. This and the sensitivity to inhibitors suggest that the site of superoxide generation at complex I is most likely the flavine mononucleotide moiety. Because short-term incubation of rat brain mitochondria with H2O2 induced increased H2O2 production at this site we propose that reactive oxygen species can activate a self-accelerating vicious cycle causing mitochondrial damage and neuronal cell death.     

Calcium pools in sea urchin eggs: roles of endoplasmic reticulum and mitochondria in relation to fertilization.

A preparation of sea urchin eggs permeabilized with digitonin (40 microM for 2.5 min) was used to study the kinetic characteristics of the two cellular compartments suspected to play a key role in cellular calcium transfer during fertilization: an ATP-dependent Ca2+ pool (Km = 0.47 microM; Vm = 0.48 nmol/min.mg protein) probably located in the endoplasmic reticulum and a mitochondrial Ca2+ pool (Km = 1.50 microM; Vm = 0.12 nmol/min.mg protein). Fertilization triggered a decrease in the rate of ATP dependent uptake by the non-mitochondrial pool (Km = 0.59 microM; Vm = 0.15 nmol/min.mg protein) while it transiently increased the Ca2+ uptake into mitochondria (2 min post-fertilization: Km = 2.20 microM; Vm = 0.40 nmol/min.mg protein). Microanalysis studies performed on quickly frozen, freeze substituted and embedded eggs showed a transient Ca2+ enrichment of mitochondria soon after fertilization thus suggesting that mitochondria behave as a Ca2+ sink at fertilization. Results are discussed in relation to the role of endoplasmic reticulum and mitochondria in handling free calcium during the early period following sea urchin egg fertilization.     

Mitochondrial fumarate reductase as a target of chemotherapy: from parasites to cancer cells

Recent research on respiratory chain of the parasitic helminth, Ascaris suum has shown that the mitochondrial NADH-fumarate reductase system (fumarate respiration), which is composed of complex I (NADH-rhodoquinone reductase), rhodoquinone and complex II (rhodoquinol-fumarate reductase) plays an important role in the anaerobic energy metabolism of adult parasites inhabiting hosts. The enzymes in these parasite-specific pathways are potential target for chemotherapy. We isolated a novel compound, nafuredin, from Aspergillus niger, which inhibits NADH-fumarate reductase in helminth mitochondria at nM order. It competes for the quinone-binding site in complex I and shows high selective toxicity to the helminth enzyme. Moreover, nafuredin exerts anthelmintic activity against Haemonchus contortus in in vivo trials with sheep indicating that mitochondrial complex I is a promising target for chemotherapy. In addition to complex I, complex II is a good target because its catalytic direction is reverse of succinate-ubiquionone reductase in the host complex II. Furthermore, we found atpenin and flutolanil strongly and specifically inhibit mitochondrial complex II. Interestingly, fumarate respiration was found not only in the parasites but also in some types of human cancer cells. Analysis of the mitochondria from the cancer cells identified an anthelminthic as a specific inhibitor of the fumarate respiration. Role of isoforms of human complex II in the hypoxic condition of cancer cells and fetal tissues is a challenge. This article is part of a Special Issue entitled Biochemistry of Mitochondria, Life and Intervention 2010.