Structural and Biochemical Studies of Substrate Selectivity in Ascaris suum Thiolases.

Thiolases are a class of carbon-carbon bond forming enzymes with important applications in biotechnology and metabolic engineering as they provide a general method for the condensation of two acyl coenzyme A (CoA) substrates. As such, developing a greater understanding of their substrate selectivity would expand our ability to engineer the enzymatic or microbial production of a broad range of small-molecule targets. Here, we report the crystal structures and biochemical characterization of Acat2 and Acat5, two biosynthetic thiolases from Ascaris suum with varying selectivity toward branched compared to linear compounds. The structure of the Acat2-C91S mutant bound to propionyl-CoA shows that the terminal methyl group of the substrate, representing the α-branch point, is directed toward the conserved Phe 288 and Met 158 residues. In Acat5, the Phe ring is rotated to accommodate a hydroxyl-π interaction with an adjacent Thr side chain, decreasing space in the binding pocket and possibly accounting for its strong preference for linear substrates compared to Acat2. Comparison of the different Acat thiolase structures shows that Met 158 is flexible, adopting alternate conformations with the side chain rotated toward or away from a covering loop at the back of the active site. Mutagenesis of residues in the covering loop in Acat5 with the corresponding residues from Acat2 allows for highly increased accommodation of branched substrates, whereas the converse mutations do not significantly affect Acat2 substrate selectivity. Our results suggest an important contribution of second-shell residues to thiolase substrate selectivity and offer insights into engineering this enzyme class.

Structural Insights into the Molecular Design of Flutolanil Derivatives Targeted for Fumarate Respiration of Parasite Mitochondria.

Recent studies on the respiratory chain of Ascaris suum showed that the mitochondrial NADH-fumarate reductase system composed of complex I, rhodoquinone and complex II plays an important role in the anaerobic energy metabolism of adult A. suum. The system is the major pathway of energy metabolism for adaptation to a hypoxic environment not only in parasitic organisms, but also in some types of human cancer cells. Thus, enzymes of the pathway are potential targets for chemotherapy. We found that flutolanil is an excellent inhibitor for A. suum complex II (IC50 = 0.058 µM) but less effectively inhibits homologous porcine complex II (IC50 = 45.9 µM). In order to account for the specificity of flutolanil to A. suum complex II from the standpoint of structural biology, we determined the crystal structures of A. suum and porcine complex IIs binding flutolanil and its derivative compounds. The structures clearly demonstrated key interactions responsible for its high specificity to A. suum complex II and enabled us to find analogue compounds, which surpass flutolanil in both potency and specificity to A. suum complex II. Structures of complex IIs binding these compounds will be helpful to accelerate structure-based drug design targeted for complex IIs.

Ukulactone C, a new NADH-fumarate reductase inhibitor produced by Talaromyces sp. FKI-6713.

Screening for NADH-fumarate reductase inhibitors led to the isolation of a new ukulactone analog, ukulactone C, as a major polyene compound produced by Talaromyces sp. FKI-6713. The structure of the compound was elucidated as a reduced analog of ukulactone A by 1D- and 2D-NMR experiments. Ukulactone C possessed a potent inhibitory activity (IC(50) = 62 nM) against NADH-fumarate reductase of the roundworm Ascaris suum in vitro.

NADH→NAD⁺ Transhydrogenation in Adult Ascaris suum Mitochondria.

Although lacking an NADPH→NAD(+) transhydrogenase system, the essentially energetically anaerobic mitochondria of the adult intestinal nematode Ascaris suum display an inner membrane-associated NADH→NAD(+) transhydrogenation reaction. This reaction is considered to be reflective of a mechanism(s) that acts in catalyzing a transmembrane translocation of reducing equivalents from NADH in the intermembrane space to matrix NAD(+), thereby forming matrix NADH that would serve in electron transport. Ascarid mitochondrial lipoamide dehydrogenase rather than an NADH→NAD(+) transhydrogenase system has been viewed as the predominant source of inner membrane-associated NADH→NAD(+) transhydrogenation activity. However, the present study made apparent yet another source of mitochondrial, inner membrane-associated NADH→NAD(+) activity in A. suum , viz., NADH dehydrogenase. This was made evident via comparisons of the A. suum mitochondrial NADH→NAD(+) transhydrogenation, NADH dehydrogenase, and lipoamide dehydrogenase activities in terms of pH effects, thermal labilities, the involvement of NADH dehydrogenase in the activities of mitochondrial, membrane-associated rotenone-insensitive and rotenone-sensitive NADH-dependent cytochrome c reductases, and mitochondrial membrane versus mitochondrial soluble localizations. Studies of the responses of the NADH→NAD(+) transhydrogenation, rotenone-insensitive and rotenone-sensitive cytochrome c reductases, and lipoamide dehydrogenase activities to inhibition by copper and cadmium lent additional support to the catalysis of an NADH→NAD(+) transhydrogenation activity by NADH dehydrogenase. Collectively, the data presented are consistent with an additional physiological catalysis of an NADH→NAD(+) transhydrogenation in A. suum mitochondria by an inner membrane NADH dehydrogenase component of the rotenone-sensitive cytochrome c reductase system, i.e., the NADH dehydrogenase component of the electron transport system. Comparisons of the A. suum data with those from other essentially anaerobic helminth parasites as well as free-living eukaryotic mitochondrial systems are noted.

Peptidases compartmentalized to the Ascaris suum intestinal lumen and apical intestinal membrane.

The nematode intestine is a tissue of interest for developing new methods of therapy and control of parasitic nematodes. However, biological details of intestinal cell functions remain obscure, as do the proteins and molecular functions located on the apical intestinal membrane (AIM), and within the intestinal lumen (IL) of nematodes. Accordingly, methods were developed to gain a comprehensive identification of peptidases that function in the intestinal tract of adult female Ascaris suum. Peptidase activity was detected in multiple fractions of the A. suum intestine under pH conditions ranging from 5.0 to 8.0. Peptidase class inhibitors were used to characterize these activities. The fractions included whole lysates, membrane enriched fractions, and physiological- and 4 molar urea-perfusates of the intestinal lumen. Concanavalin A (ConA) was confirmed to bind to the AIM, and intestinal proteins affinity isolated on ConA-beads were compared to proteins from membrane and perfusate fractions by mass spectrometry. Twenty-nine predicted peptidases were identified including aspartic, cysteine, and serine peptidases, and an unexpectedly high number (16) of metallopeptidases. Many of these proteins co-localized to multiple fractions, providing independent support for localization to specific intestinal compartments, including the IL and AIM. This unique perfusion model produced the most comprehensive view of likely digestive peptidases that function in these intestinal compartments of A. suum, or any nematode. This model offers a means to directly determine functions of these proteins in the A. suum intestine and, more generally, deduce the wide array functions that exist in these cellular compartments of the nematode intestine.

Molecular cloning and analysis of Ancylostoma ceylanicum glutamate-cysteine ligase.

Glutamate-cysteine ligase (GCL) is a heterodimer enzyme composed of a catalytic subunit (GCLC) and a modifier subunit (GCLM). This enzyme catalyses the synthesis of γ-glutamylcysteine, a precursor of glutathione. cDNAs of the putative glutamate-cysteine ligase catalytic (Ace-GCLC) and modifier subunits (Ace-GCLM) of Ancylostoma ceylanicum were cloned using the RACE-PCR amplification method. The Ace-gclc and Ace-gclm cDNAs encode proteins with 655 and 254 amino acids and calculated molecular masses of 74.76 and 28.51kDa, respectively. The Ace-GCLC amino acid sequence shares about 70% identity and 80% sequence similarity with orthologs in Loa loa, Onchocerca volvulus, Brugia malayi, and Ascaris suum, whereas the Ace-GCLM amino acid sequence has only about 30% sequence identity and 50% similarity to homologous proteins in those species. Real-time PCR analysis of mRNA expression in L3, serum stimulated L3 and adult stages of A. ceylanicum showed the highest level of Ace-GCLC and Ace-GCLM expression occurred in adult worms. No differences were detected among adult hookworms harvested 21 and 35dpi indicating expression of Ace-gclc and Ace-gclm in adult worms is constant during the course of infection. Positive interaction between two subunits of glutamate-cysteine ligase was detected using the yeast two-hybrid system, and by specific enzymatic reaction. Ace-GCL is an intracellular enzyme and is not exposed to the host immune system. Thus, as expected, we did not detect IgG antibodies against Ace-GCLC or Ace-GCLM on days 21, 60 and 120 of A. ceylanicum infection in hamsters. Furthermore, vaccination with one or both antigens did not reduce worm burdens, and resulted in no improvement of clinical parameters (hematocrit and hemoglobin) of infected hamsters. Therefore, due to the significant role of the enzyme in parasite metabolism, our analyses raises hope for the development of a successful new drug against ancylostomiasis based on the specific GCL inhibitor.

Diversity of parasite complex II.

Parasites have developed a variety of physiological functions necessary for completing at least part of their life cycles in the specialized environments of surrounding the parasites in the host. Regarding energy metabolism, which is essential for survival, parasites adapt to the low oxygen environment in mammalian hosts by using metabolic systems that are very different from those of the hosts. In many cases, the parasite employs aerobic metabolism during the free-living stage outside the host but undergoes major changes in developmental control and environmental adaptation to switch to anaerobic energy metabolism. Parasite mitochondria play diverse roles in their energy metabolism, and in recent studies of the parasitic nematode, Ascaris suum, the mitochondrial complex II plays an important role in anaerobic energy metabolism of parasites inhabiting hosts by acting as a quinol-fumarate reductase. In Trypanosomes, parasite complex II has been found to have a novel function and structure. Complex II of Trypanosoma cruzi is an unusual supramolecular complex with a heterodimeric iron-sulfur subunit and seven additional non-catalytic subunits. The enzyme shows reduced binding affinities for both substrates and inhibitors. Interestingly, this structural organization is conserved in all trypanosomatids. Since the properties of complex II differ across a wide range of parasites, this complex is a potential target for the development of new chemotherapeutic agents. In this regard, structural information on the target enzyme is essential for the molecular design of drugs. This article is part of a Special Issue entitled: Respiratory complex II: Role in cellular physiology and disease.

Purification and partial biochemical-genetic characterization of trehalose 6-phosphate synthase from muscles of adult female Ascaris suum.

Trehalose 6-phosphate (T6P) synthase (TPS; EC 2.4.1.15) was isolated from muscles of Ascaris suum by ammonium sulphate fractionation, ion-exchange DEAE SEPHACEL(TM) anion exchanger column chromatography and Sepharose 6B gel filtration. On sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), 265-fold purified TPS exhibited a molecular weight of 66 kDa. The optimum pH and temperature of the purified enzyme were 3.8-4.2 and 35°C, respectively. The isoelectric point (pI) of TPS was pH 5.4. The studied TPS was not absolutely substrate specific. Besides glucose 6-phosphate, the enzyme was able to use fructose 6-phosphate as an acceptor of glucose. TPS was activated by 10 mM MgCl2, 10 mM CaCl2 and 10 mM NaCl. In addition, it was inhibited by ethylenediaminetetra-acetic acid (EDTA), KCl, FeCl3 and ZnCl2. Two genes encoding TPS were isolated and sequenced from muscles of the parasite. Complete coding sequences for tps1 (JF412033.2) and tps2 (JF412034.2) were 3917 bp and 3976 bp, respectively. Translation products (AEX60788.1 and AEX60787.1) showed expression to the glucosyltransferase-GTB-type superfamily.

Synthesis of N²-modified 7-methylguanosine 5'-monophosphates as nematode translation inhibitors.

Preparative scale synthesis of 14 new N(2)-modified mononucleotide 5' mRNA cap analogues was achieved. The key step involved use of an S(N)Ar reaction with protected 2-fluoro inosine and various primary and secondary amines. The derivatives were tested in a parasitic nematode, Ascaris suum, cell-free system as translation inhibitors. The most effective compound with IC(50) ∼0.9µM was a N(2)-p-metoxybenzyl-7-methylguanosine-5'-monophosphate 35.

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.

Ascaris suum enolase is a potential vaccine candidate against ascariasis.

Ascariasis caused by Ascaris is the most common parasite problem in humans and pigs worldwide. No vaccines are available for the prevention of Ascaris infections. In the present study, the gene encoding Ascaris suum enolase (As-enol-1) was amplified, cloned and sequenced. Amino acid sequence alignment indicated that As-enol-1 was highly conserved between different nematodes and shared the highest identity (87%) with enolase from Anisakis simplex s.l. The recombinant pVAX-Enol was successfully expressed in Marc-145 cells. The ability of the pVAX-Enol for inducing immune protective responses against challenge infection with A. suum L3 was evaluated in Kunming mice. The immune response was evaluated by lymphoproliferative assay, cytokine and antibody measurements, and the reduction rate of recovery larvae. The results showed that the mice immunized with pVAX-Enol developed a high level of specific antibody responses against A. suum, a strong lymphoproliferative response, and significant levels of IFN-γ, IL-2, IL-4 and IL-10 production, compared with the other groups immunized with empty plasmid or blank controls, respectively. There was a 61.13% reduction (P<0.05) in larvae recovery compared with that in the blank control group. Our data indicated that A. suum enolase is a potential vaccine candidate against A. suum infection.

Molecular and catalytic properties of an arginine kinase from the nematode Ascaris suum.

We amplified the cDNA coding for arginine kinase (AK) from the parasitic nematode Ascaris suum, cloned it in pMAL plasmid and expressed the enzyme as a fusion protein with the maltose-binding protein. The whole cDNA was 1260 bp, encoding 400 amino acids, and the recombinant protein had a molecular mass of 45,341 Da. Ascaris suum recombinant AK showed significant activity and strong affinity ( K(m)(Arg) = 0.126 mM) for the substrate L-arginine. It also exhibited high catalytic efficiency ( k(ca)/K(m)(Arg) = 352) comparable with AKs from other organisms. Sequence analysis revealed high amino acid sequence identity between A. suum AK and other nematode AKs, all of which cluster in a phylogenetic tree. However, comparison of gene structures showed that A. suum AK gene intron/exon organization is quite distinct from that of other nematode AKs. Phosphagen kinases (PKs) from certain parasites have been shown to be potential novel drug targets or tools for detection of infection. The characterization of A. suum AK will be useful in the development of strategies for control not only of A. suum but also of related species infecting humans.

Ascaris suum: RNAi mediated silencing of enolase gene expression in infective larvae.

Ascaris suum is an important parasite of pigs that causes tremendous economic losses globally to agriculture and animal husbandry annually. RNA interference (RNAi) technology has been described as a successful and useful approach for the elucidation of gene function in parasitic nematodes. In the present study, RNAi was used to silence the expression of a gene encoding enolase in A. suum by soaking infective larvae in double-stranded RNA derived from an EST (representing As-enol-1) selected from an A. suum infective larvae-specific cDNA library. The mRNA levels of RNAi-treated larvae were examined by Reverse-Transcription PCR (RT-PCR) analysis. The survival of RNAi-treated larvae was compared with larvae treated with dsRNA-free culture medium. The effect of enolase depletion on the development of A. suum larvae was assessed by infecting BALB/c mice with RNAi-treated larvae. The results showed that enolase gene expression was silenced completely and the survival rate of the RNAi-treated nematodes was reduced by 20.11% (P<0.01) after soaking for 72 h. Although no significant difference was detected in the numbers of larvae recovered from the liver and lungs of infected mice 4 days post infection, RNAi knockdown of the A. suum enolase mRNA led to significant shorter larvae, indicating that loss of enolase expression may cause delays in larval development.

Crystallization of mitochondrial rhodoquinol-fumarate reductase from the parasitic nematode Ascaris suum with the specific inhibitor flutolanil.

In adult Ascaris suum (roundworm) mitochondrial membrane-bound complex II acts as a rhodoquinol-fumarate reductase, which is the reverse reaction to that of mammalian complex II (succinate-ubiquinone reductase). The adult A. suum rhodoquinol-fumarate reductase was crystallized in the presence of octaethyleneglycol monododecyl ether and n-dodecyl-beta-D-maltopyranoside in a 3:2 weight ratio. The crystals belonged to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 123.75, b = 129.08, c = 221.12 A, and diffracted to 2.8 A resolution using synchrotron radiation. The presence of two molecules in the asymmetric unit (120 kDa x 2) gives a crystal volume per protein mass (V(M)) of 3.6 A(3) Da(-1).

Ascaris suum NADH-methemo(myo)globin reductase systems recovering differential functions of hemoglobin and myoglobin, adapting to environmental hypoxia.

We reported previously that Ascaris suum cytochrome b5, specifically expressed in this nematode at the adult stage and dually localized in extracellular perienteric fluid and hypodermis, is involved in both perienteric NADH-methemoglobin and cytosolic NADH-metmyoglobin reduction, where cytochrome b5 functions as an electron carrier between NADH-mediated cytochrome b5 reductase and substrates, methemo(myo)globins to reduce the nonfunctional globins back to functional ferrous hemo(myo)globins. To further characterize NADH-methemo(myo)globin reductase systems, the midpoint potentials of A. suum perienteric hemoglobin and body wall myoglobin, as well as the affinities of Ascaris methemoglobin and metmyoglobin toward cytochrome b5, were evaluated using potentiometric titration and surface plasmon resonance techniques, respectively. Midpoint potentials of +7.2 mV and +19.5 mV were obtained for Ascaris perienteric hemoglobin and body wall myoglobin, respectively. The affinities of Ascaris perienteric methemoglobin and body wall metmyoglobin toward the nematode cytochrome b5 were comparable to that for mammalian hemoglobin and cytochrome b5; association constants were 0.585 x 10(3) M(-1) and 2.32 x 10(3) M(-1), respectively, with rapid equilibration kinetics. These observations highlight the physiological importance of A. suum perienteric NADH-methemoglobin and cytosolic metmyoglobin reductase systems. Differential roles of A. suum perienteric hemoglobin and body wall myoglobin are also discussed from the viewpoint of oxygen homeostasis under hypoxic conditions.

Role of residues in the adenosine binding site of NAD of the Ascaris suum malic enzyme.

Ascaris suum mitochondrial malic enzyme catalyzes the divalent metal ion dependent conversion of l-malate to pyruvate and CO(2), with concomitant reduction of NAD(P) to NAD(P)H. In this study, some of the residues that form the adenosine binding site of NAD were mutated to determine their role in binding of the cofactor and/or catalysis. D361, which is completely conserved among species, is located in the dinucleotide-binding Rossmann fold and makes a salt bridge with R370, which is also highly conserved. D361 was mutated to E, A and N. R370 was mutated to K and A. D361E and A mutant enzymes were inactive, likely a result of the increase in the volume in the case of the D361E mutant enzyme that caused clashes with the surrounding residues, and loss of the ionic interaction between D361 and R370, for D361A. Although the K(m) for the substrates and isotope effect values did not show significant changes for the D361N mutant enzyme, V/E(t) decreased by 1400-fold. Data suggested the nonproductive binding of the cofactor, giving a low fraction of active enzyme. The R370K mutant enzyme did not show any significant changes in the kinetic parameters, while the R370A mutant enzyme gave a slight change in V/E(t), contrary to expectations. Overall, results suggest that the salt bridge between D361 and R370 is important for maintaining the productive conformation of the NAD binding site. Mutation of residues involved leads to nonproductive binding of NAD. The interaction stabilizes one of the Rossmann fold loops that NAD binds. Mutation of H377 to lysine, which is conserved in NADP-specific malic enzymes and proposed to be a cofactor specificity determinant, did not cause a shift in cofactor specificity of the Ascaris malic enzyme from NAD to NADP. However, it is confirmed that this residue is an important second layer residue that affects the packing of the first layer residues that directly interact with the cofactor.

Proper positioning of the nicotinamide ring is crucial for the Ascaris suum malic enzyme reaction.

The mitochondrial NAD-malic enzyme catalyzes the oxidative decarboxylation of malate to pyruvate and CO2. The role of the dinucleotide substrate in oxidative decarboxylation is probed in this study using site-directed mutagenesis to change key residues that line the dinucleotide binding site. Mutant enzymes were characterized using initial rate kinetics, and isotope effects were used to obtain information on the contribution of these residues to binding energy and catalysis. Results obtained for the N479 mutant enzymes indicate that the hydrogen bond donated by N479 to the carboxamide side chain of the nicotinamide ring is important for proper orientation in the hydride transfer step. The stepwise oxidative decarboxylation mechanism observed for the wt enzyme changed to a concerted one, which is totally rate limiting, for the N479Q mutant enzyme. In this case, it is likely that the longer glutamine side chain causes reorientation of malate such that it binds in a conformation that is optimal for concerted oxidative decarboxylation. Converting N479 to the shorter serine side chain gives very similar values of KNAD, Kmalate, and isotope effects relative to wt, but V/Et is decreased 2 000-fold. Data suggest an increased freedom of rotation, resulting in nonproductively bound cofactor. Changes were also made to two residues, S433 and N434, which interact with the nicotinamide ribose of NAD. In addition, N434 donates a hydrogen bond to the beta-carboxylate of malate. The KNAD for the S433A mutant enzyme increased by 80-fold, indicating that this residue provides significant binding affinity for the dinucleotide. With N434A, the interaction of the residue with malate is lost, causing the malate to reorient itself, leading to a slower decarboxylation step. The longer glutamine and methionine side chains stick into the active site and cause a change in the position of malate and/or NAD resulting in more than a 104-fold decrease in V/Et for these mutant enzymes. Overall, data indicate that subtle changes in the orientation of the cofactor and substrate dramatically influence the reaction rate.

Ascaris suum cytochrome b5, an adult-specific secretory protein reducing oxygen-avid ferric hemoglobin.

The anaerobic parasitic nematode Ascaris suum has an oxygen-avid hemoglobin in the perienteric fluid, the biological function of which remains elusive. Here, we report that Ascaris cytochrome b5 is expressed specifically in the intestinal parasitic stage and is secreted into the perienteric fluid, thus co-localizing with Ascaris hemoglobin. We also found that cytochrome b5 reduces Ascaris non-functioning ferric methemoglobin more efficiently than mammalian methemoglobin. Furthermore, a computer graphics model of the electron transfer complex between Ascaris cytochrome b5 and Ascaris hemoglobin strongly suggested that these two proteins are physiological redox partners. Nitric oxide has been reported to react easily with oxygen captured in hemoglobin to form nitrate, but not toxic free radicals, which may result in production of methemoglobin for the cytochrome b5 to regenerate functional ferrous hemoglobin. Therefore, our findings suggest that Ascaris cytochrome b5 is a key redox partner of Ascaris hemoglobin, which acts as an antioxidant.

Change of subunit composition of mitochondrial complex II (succinate-ubiquinone reductase/quinol-fumarate reductase) in Ascaris suum during the migration in the experimental host.

The mitochondrial metabolic pathway of the parasitic nematode Ascaris suum changes dramatically during its life cycle, to adapt to changes in the environmental oxygen concentration. We previously showed that A. suum mitochondria express stage-specific isoforms of complex II (succinate-ubiquinone reductase: SQR/quinol-fumarate reductase: QFR). The flavoprotein (Fp) and small subunit of cytochrome b (CybS) in adult complex II differ from those of infective third stage larval (L3) complex II. However, there is no difference in the iron-sulfur cluster (Ip) or the large subunit of cytochrome b (CybL) between adult and L3 isoforms of complex II. In the present study, to clarify the changes that occur in the respiratory chain of A. suum larvae during their migration in the host, we examined enzymatic activity, quinone content and complex II subunit composition in mitochondria of lung stage L3 (LL3) A. suum larvae. LL3 mitochondria showed higher QFR activity ( approximately 160 nmol/min/mg) than mitochondria of A. suum at other stages (L3: approximately 80 nmol/min/mg; adult: approximately 70 nmol/min/mg). Ubiquinone content in LL3 mitochondria was more abundant than rhodoquinone ( approximately 1.8 nmol/mg versus approximately 0.9 nmol/mg). Interestingly, the results of two-dimensional bule-native/sodium dodecyl sulfate polyacrylamide gel electrophoresis analyses showed that LL3 mitochondria contained larval Fp (Fp(L)) and adult Fp (Fp(A)) at a ratio of 1:0.56, and that most LL3 CybS subunits were of the adult form (CybS(A)). This clearly indicates that the rearrangement of complex II begins with a change in the isoform of the anchor CybS subunit, followed by a similar change in the Fp subunit.