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Cyanide-insensitive trypanosome alternative oxidase (TAO) is the terminal oxidase of the respiratory chain of long slender bloodstream forms of the African trypanosome, which causes sleeping sickness in humans and nagana in cattle. TAO has been targeted for the development of anti-trypanosomal drugs, because it does not exist in the host. In this study, we established a system for overproduction of highly active TAO in Eschericia coli heme-deficient mutant. Kinetic analysis of recombinant enzyme and TAO in Trypanosoma brucei brucei mitochondria revealed that recombinant TAO retains the properties of native enzyme, indicating that recombinant TAO is quite valuable for further biochemical study of TAO.

The heme molecule plays an important role in the electron transfer performed by redox proteins such as cytochromes. In addition, it has been suggested that heme has a structural role in protein folding and assembly. However, it is now known that protein folding and assembly do not occur spontaneously in vivo, but rather, a series of helper proteins, known as molecular chaperones, are required. Thus, in this article, we show the role of heme and various molecular chaperones in the in vivo assembly of a tetrameric membrane protein called succinate-ubiquinone reductase (SQR, complex II), which contains heme b and functions as a dehydrogenase in the aerobic respiratory chain, in Escherichia coli.

Parasites have developed a variety of physiological functions necessary for their survival within the specialized environment of the host. Using metabolic systems that are very different from those of the host, they can adapt to low oxygen tension present within the host animals. Most parasites do not use the oxygen available within the host to generate ATP, but rather employ systems anaerobic metabolic pathways. The enzymes in these parasite-specific pathways are potential targets for chemotherapy.Cyanide-insensitive trypanosome alternative oxidase (TAO) is the terminal oxidase of the respiratory chain of long slender bloodstream forms of the African trypanosome, which causes sleeping sickness in human and nagana in cattle. TAO has been targeted for the development of anti-trypanosomal drugs because it does not exist in the host. Recently, we found the most potent inhibitor of TAO to date, ascofuranone, a compound isolated from the phytopathogenic fungus, Ascochyta visiae.

Cyanide-insensitive trypanosome alternative oxidase (TAO) is the terminal oxidase of the respiratory chain of long slender bloodstream forms of the African trypanosome, which causes sleeping sickness in human and nagana in cattle. TAO has been targeted for the development of anti-trypanosomal drugs because it does not exist in the host. The cDNA for TAO has been cloned from Trypanosoma brucei brucei EATRO110 strain and has been used for further characterization. In this study, we found amino acid sequence of the C-terminal part of TAO from the strain that we are using, T. b. brucei TC221, is considerably different from that of the EATRO110 strain.

Heme molecules play important roles in electron transfer by redox proteins such as cytochromes. In addition, a structural role for heme in protein folding and the assembly of enzymes has been suggested. Previous results obtained using Escherichia coli hemA mutants, which are unable to synthesize 5-aminolevulinic acid, a precursor of porphyrins and hemes, have demonstrated a requirement for heme biosynthesis in the assembly of a functional succinate-ubiquinone reductase (SQR or complex II), which is a component of the aerobic respiratory chain. In the present study, in order to investigate the role of the heme in the assembly of E. coli SQR, we used a hemH (encodes ferrochelatase) mutant that lacks the ability to insert iron into the porphyrin ring. The hemH mutant failed to insert functional SQR into the cytoplasmic membrane, and the catalytic portion of SQR [the flavoprotein subunit (Fp) and the iron-sulfur protein subunit (Ip)] was localized in the cytoplasm of the cell. It is of interest to note that protoporphyrin IX accumulated in the mutant cells and inactivated the cytoplasmic succinate dehydrogenase (SDH) activity associated with the catalytic Fp-Ip complex. In contrast, SQR was assembled into the membrane of a heme-permeable hemH double mutant when hemin was present in the culture. Only a low level of SQR activity was found in the membrane when hemin was replaced by non-iron metalloporphyrins: Mn-, Co-, Ni-, Zn- and Cu-protoporphyrin IX, or protoporphyrin IX These results indicate that heme iron is indispensable for the functional assembly of SQR in the cytoplasmic membrane of E. coli, and provide a new insight into the biological role of heme in the molecular assembly of the multi-subunit enzyme complex.

Parasites have developed a wide variety of physiological functions to survive within the specialized environments of the host. Regarding energy metabolism, which represents an essential factor for survival, parasites adapt low oxygen tension in host mammals using metabolic systems that differ substantially from those of the host. Most parasites do not use free oxygen available within the host, but employ systems other than oxidative phosphorylation for ATP synthesis. Furthermore, parasites display marked changes in mitochondrial morphology and components during the life cycle, and these represent very interesting elements of biological processes such as developmental control and environmental adaptation. The enzymes in parasite-specific pathways offer potential targets for chemotherapy. Cyanide-insensitive trypanosome alternative oxidase (TAO) is the terminal oxidase of the respiratory chain of long slender bloodstream forms of the African trypanosome, which causes sleeping sickness. Recently, the most potent inhibitor of TAO to date, ascofuranone, was isolated from the phytopathogenic fungus, Ascochyta visiae. The inhibitory mechanisms of ascofuranone have been revealed using recombinant enzyme. Parasite-specific respiratory systems are also found in helminths. The NADH-fumarate reductase system in mitochondria form a final step in the phosphoenolpyruvate carboxykinase (PEPCK)-succinate pathway, which plays an important role in anaerobic energy metabolism for the Ascaris suum adult. Enzymes in this system, such as NADH-rhodoquinone reductase (complex I) and rhodoquinol-fumarate reductase (complex II), form promising targets for chemotherapy. In fact, a specific inhibitor of nematode complex I, nafuredin, has been found in mass-screening using parasite mitochondria.

Trypanosome alternative oxidase (TAO) is the terminal oxidase of the respiratory chain in long slender bloodstream forms of African trypanosomes. TAO is a cytochrome-independent, cyanide-insensitive quinol oxidase. These characteristics are distinct from those of the bacterial quinol oxidases, proteins that belong to the heme-copper terminal oxidase superfamily. The inability to purify stable TAO has severely hampered biochemical studies of the alternative oxidase family. In the present study, we were able to purify recombinant TAO to homogeneity from Escherichia coli membranes using the detergent digitonin. Kinetic analysis of the purified TAO revealed that the specific inhibitor ascofuranone is a competitive inhibitor of ubiquinol oxidase activity.