Kiyoshi Kita

Developmental changes in the respiratory chain of Ascaris mitochondria

The Ascaris larval respiratory chain, particularly complex II (succinate-ubiquinone oxidoreductase), was characterized in isolated mitochondria. Low-temperature difference spectra showed the presence of substrate-reducible cytochromes aa3 of complex IV, c+c1 and b of complex III (ubiquinol-cytochrome c oxidoreductase) in mitochondria from second-stage larvae (L2 mitochondria). Quinone analysis by high-performance liquid chromatography showed that, unlike adult mitochondria, which contain only rhodoquinone-9, L2 mitochondria contain ubiquinone-9 as a major component. Complex II in L2 mitochondria was kinetically different from that in adult mitochondria. The individual oxidoreductase activities comprising succinate oxidase, and fumarate reductase were determined in mitochondria from L2 larvae, from larvae cultured to later stages, and from adult nematodes. The L2 mitochondria exhibited the highest specific activity of cytochrome c oxidase, indicating that L2 larvae have the most aerobic respiratory chain among the stages studied. The Cybs subunit of complex II in L2 and cultured-larvae mitochondria exhibited different reactivities against anti-adult Cybs antibodies. Taken together, these results indicate that the complex II of larvae is different from its adult counterpart. In parallel with this change in mitochondrial biogenesis, biosynthetic conversion of quinones occurs during development in Ascaris nematodes.

Electron-transfer complexes of Ascaris suum muscle mitochondria. III. Composition and fumarate reductase activity of complex II

Complex II of the anaerobic respiratory chain in Ascaris muscle mitochondria showed a high fumarate reductase activity when reduced methyl viologen was used as the electron donor. The maximum activity was 49 mumol/min per mg protein, which is much higher than that of the mammalian counterpart. The mitochondria of Ascaris-fertilized eggs, which require oxygen for its development, also showed fumarate reductase activity with a specific activity intermediate between those of adult Ascaris and mammals. Antibody against the Ascaris flavoprotein subunit reacted with the mammalian counterparts, whereas those against the Ascaris iron-sulfur protein subunit did not crossreact, although the amino acid compositions of the subunits in Ascaris and bovine heart were quite similar. Cytochrome b-558 of Ascaris complex II was separated from flavoprotein and iron-sulphur protein subunits by high performance liquid chromatography with a gel permeation system in the presence of Sarkosyl. Isolated cytochrome b-558 is composed of two hydrophobic polypeptides with molecular masses of 17.2 and 12.5 kDa determined by gradient gel, which correspond to the two small subunits of complex II. Amino acid compositions of these small subunits showed little similarity with those of cytochrome b-560 of bovine heart complex II. NADH-fumarate reductase, which is the final enzyme complex in the anaerobic respiratory chain in Ascaris, was reconstituted with bovine heart complex I, Ascaris complex II and phospholipids. The maximum activity was 430 nmol/min per mg protein of complex II. Rhodoquinone was essential for this reconstitution, whereas ubiquinone showed no effect. The results clearly indicate the unique role of Ascaris complex II as fumarate reductase and the indispensability of rhodoquinone as the low-potential electron carrier in the NADH-fumarate reductase system.

Human complex II (succinate-ubiquinone oxidoreductase) : cDNA cloning of iron sulfur (Ip) subunit of liver mitochondria

Complex II (succinate-ubiquinone oxidoreductase) is an important enzyme complex of both the tricarboxylic acid cycle and of the aerobic respiratory chains of mitochondria in eukaryotic cell and prokaryotic organisms. In this study, the amino acid sequence of iron sulfur-subunit in human liver mitochondria was deduced from cDNA which was isolated by immunoscreening a human liver lambda gtll cDNA library. An isolated clone contains an open reading frame of 786 nucleotides and encodes a mature protein of 252 amino acids with a molecular weight of 28,804. The amino acid sequence was highly homologous with that of bovine heart (94.1%) which has been determined from the purified peptide and that of Escherichia coli sdh B product (50.8%). Striking sequence conservation was found around the three cysteine-rich clusters which have been thought to comprise the iron-sulfur centers of the enzyme. This is the first report on the cDNA sequence of mitochondrial complex II.

Complex II is a major component of the respiratory chain in the muscle mitochondria of Ascaris suum with high fumarate reductase activity

1. 1. Analysis with sodium dodecyl sulfate polyacrylamide gel electrophoresis and High Performance Liquid Chromatography in the presence of detergent showed that complex II (succinate—ubiquinone reductase) is one of the major components of mitochondria in Ascaris suum muscle, and the content of complex II in Ascaris muscle mitochondria is quite high, compared with that of rat liver. 2. 2. Most of the b cytochrome in Ascaris muscle mitochondria was identified to be cytochrome b558 in complex II. 3. 3. Complex II of Ascaris mitochondria showed high fumarate reductase activity when the activity was measured using methyl viologen as an electron donor.

ESR studies on iron-sulfur clusters of complex II in Ascaris suum mitochondria which exhibits strong fumarate reductase activity

Complex II of Ascaris suum mitochondria, which functions as fumarate reductase in physiological conditions, contains three types of iron‐sulfur clusters. These correspond to clusters S‐1, S‐2 and S‐3 and are distinguishable by low‐temperature ESR studies. Cluster S‐1 is reduced by succinate, giving ESR signals with g z, g y and g x values at 2.033, 1.939 and 1.920. The existence of cluster S‐2 is suggested by an enhancement of the S‐1 spin relaxation induced upon reduction of S‐2 by dithionite. Cluster S‐3 is ESR detectable under air‐oxidized conditions and gives a strong signal at g= 2.025. Cluster S‐3 was only partially reduced even with an excess amount of sodium succinate, which is a common characteristic of fumarate reductase but this is not seen in the mitochondrial complex II.

Structural studies on three flavin-interacting regions of the flavoprotein subunit of complex II in Ascaris suum mitochondria.

The flavoprotein (Fp) subunit of mitochondrial complex II contains covalently bound FAD as a prosthetic group. In this study, the primary structure of the flavin‐bound tryptic peptide from the Fp subunit of Ascaris complex II was determined and found to be highly similar to those of the corresponding flavin‐binding regions of bovine heart and bacterial Fp subunits. Furthermore, the Ascaris Fp subunit was shown to contain two regions exhibiting striking sequence similarity to the segments that have been predicted to interact noncovalently with the AMP moiety of FAD in bacterial Fp subunits. The conservation of these two regions also in themitochondrial Fp subunit suggests their functional importance.

The Physiological Significance of Complex II (Succinate-Ubiquinone Reductase) in Respiratory Adaptation

Several features of the molecular assembly of the fumarate reductase system of some parasitic helminths have been characterised. Cytochrome b558 participates in the NADH-fumarate reductase system in muscle mitochondria of Ascaris adult worms. The cytochrome b was reduced with various substrates and the reduced cytochrome was oxidised by fumarate in both preparations of mitochondria and mitochondrial membrane fragments. Similar cytochromes b were present in the other parasitic helminths in which the PEPCK-succinate pathway is operative.

Chromosomal location of the Escherichia coli cytochrome b556 gene, cybA

SummaryThe amounts of cytochrome b556 in the cytoplasmic membranes of several Escherichia coli K12 strains having F-prime factors and a lambda transducing phage were determined. The amount was amplified about two-fold in strains having F100-12 and F152, but not in strains having F100-11, F8 and λpsu+2glnS+. The strain TK3D11, which lacks the kdp-gltA region (deletion D-01) of the E. coli chromosome, did not synthesize cytochrome b556 at all. From these results, the gene cybA encoding cytochrome b556 was located in the kdp-gltA region.In the cytochrome b556-deficient mutant, a novel b type cytochrome, cytochrome b561 which is a product of the gene cybB, was identified. It seems to function as a physiological electron transferring cytochrome in place of cytochrome b556 in this mutant.

Properties of Cytochrome b558 in Complex II of Ascaris Muscle Mitochondria and its Function in Anaerobic Respiration

The phosphoenolpyruvate carboxykinase (PEPCK)-succinate pathway is important in the energy metabolism of anaerobic organisms such as Ascaris suum, which resides in the host’s small intestine where oxygen is fairly limited (1). The last step of this pathway is catalized by fumarate reductase activity of mitochondria, and our studies on the respiratory chain of mitochondria from Ascaris indicated that complex II (succinate-ubiquinone reductase) functions as fumarate reductase by its reverse reaction. Complex II contains b-type cytochrome and properties of this cytochrome of Ascaris was somewhat different from that of beef heart. Comparative characterization of these b cytochromes are described.