Electron transport chain

A continuous supply of energy in the form of ATP is essential to the maintenance of life. In most eukaryotes, it is achieved by oxygen-dependent energy production and the mitochondrial electron transport chain plays a central role in ATP production. In higher eukaryotes, the electron transport chain comprises four integral membrane protein complexes namely, NADH:ubiquinone oxidoreductase (complex I), succinate:ubiquinone oxidoreductase (complex II), ubiquinol:cytochrome c oxidoreductase/ cytochrome bc1 complex (complex III) and cytochrome oxidase (complex IV).  The electrons are transferred from NADH and succinate to oxygen through these series of enzymatic complexes of the inner mitochondrial membrane and oxygen is reduced to water. This releases energy and generates a proton gradient across mitochondrial membrane by pumping protons into the intermembrane space. The energy of oxidation of hydrogen is used to phosphorylate ADP into ATP. This ATP generation is catalysed by ATP synthase complex (complex V).

The conventional NADH:ubiquinone oxidoreductase multiprotein complex is absent in the genomes of both Plasmodium falciparum and Toxoplasma gondii. However, an alternative single gene NAD(P)H dehydrogenase enzyme homologous to the peripheral membrane NADH dehydrogenase in yeast, plants and fungi was identified in P. falciparum genome. These alternative NADH dehydrogenases of mitochondrial membrane are insensitive to rotenone, an inhibitor of complex I [1]. The presence of oxidation of exogenous NADH and its insensitivity to rotenone has been experimentally shown in P. falciparum and P. yoelii yoelii. This confirms the presence of alternative NADH dehydrogenase in these species [2, 3, 4]. The analysis of T. gondii genome has showed that there are two NAD(P)H dehydrogenases that are orthologous to the Plasmodium alternative NAD(P)H dehydrogenase. The inhibition of T. gondii NAD(P)H dehydrogenase with 1-hydroxy-2-dodecyl-4(1H)quinolone has led to the collapse of mitochondrial inner membrane potential and depletion of ATP [5] suggesting the importance of this enzyme in mitochondrial electron transport and its role in ATP production. In addition, studies have suggested that both the isoforms of NADH dehydrogenase are essential for the growth of T. gondii and even single knockout mutants displayed a decreased replication rate and also decreased mitochondrial membrane potential [6].

The analysis of the genome of T. gondii shows the presence of only two subunits of succinate dehydrogenase (flavoprotein subunit and iron-sulphur protein subunits). The orthologs of the membrane anchor subunits have not been identified in the T. gondii genome. It was also the case with P. falciparum genome. Purification and molecular characterisation of the succinate dehydrogenase in P. falciparum has identified the above mentioned two subunits (flavoprotein subunit and iron-sulphur protein subunits)  and demonstrated significant activity. It is also suggested that this enzyme may be a peripheral complex [4, 7, 8]. The complex III of P. falciparum and T. gondii are similar to mammalian enzymes. The mammalian complex III inhibitors such as myxothiazol and antimycin A can inhibit Plasmodium complex III activity [4]. The differences in the ubiquinol binding regions of Plasmodium cytochrome b to the mammalian protein has enabled the use of atovaquone as anti-malarial. Its action in complex III was confirmed by the study showing the resistance to atovoquone in malarial parasites with mutations in the ubiquinol binding domains of cytochrome b [9]. The use of related drugs to treat East coast fever, an infection of another apicomplexan, Theileria parva [10] may suggest the conservation of ubiquinol binding domain of cytochrome b across all apicomplexan species including Toxoplasma.

The analysis of Toxoplasma genome has led to identification of two subunits of Cox2, the accessory protein Cox4 and the assembly proteins Cox10, Cox11, Cox12, Cox15, Cox17 and Cox19. The orthologs of all these genes are present in the P. falciparum genome (PlasmoDB/MPMP). The Cox1 and Cox3 are present in the mitochondrial genome which has been reported in P. falciparum and P. yoelii [11, 12] . The Plasmodium mitochondrial genome available in PlasmoDB also possess the genes for Cox1 and Cox3 and reconstructed in MPMP pathway. The activity of Cox complex has been evident from the study in the malaria parasite P. berghei [13].

The genomes of both P. falciparum and T. gondii possess the genes for all the F₁ subunits and the F_o-c subunit (proteolipid subunit). The genes for F_o-a and F_o-b are not identified in these species [4, 10]. It has been demonstrated in intra-erythrocytic stages of P. falciparum that electron transport chain does not play a role in ATP synthesis and it is only important for regeneration of ubiquinone as an electron acceptor for dihydroorotate dehydrogenase. It has also been proposed that the hydrolysis of ATP by matrix localised ATP synthase and transport of ADP for ATP will generate net negative charge and establishes membrane potential [14]. Although F_o-a and F_o-b subunits of ATP synthase are also missing in T. gondii gene models, it has been demonstrated that electron transport chain mediated generation of ATP is essential for T. gondii. The 30% reduction of ATP levels with inhibition of NAD(P)H dehydrogenase with 1-hydroxy-2-dodecyl-4(1H)quinolone within one hour and 70% reduction within 24 hours, 70% reduction in ATP levels with F_o subunit inhibitor oligomycin and oligomycin leading to stabilisation of membrane potential in the presence of 1-hydroxy-2-dodecyl-4(1H)quinolone suggests the importance of F_o subunits of ATP synthase. This also suggests the importance of electron transport and oxidative phosphorylation in ATP generation in T. gondii. These differences  in electron transport chain might have been due to the differences in life styles of Plasmodium and Toxoplasma [5]. In addition, the mitochondrial respiratory chain has also been proved to be essential in Plasmodium yoeli as the inhibition of electron transport and mitochondrial depolarization with atovaquone treatment caused cellular damage and death [15]. The same effect was also observed with atovaquone in T. gondii [16]. All these evidence suggest that the respiratory chain and oxidative phosphorylation are functional in apicomplexans T. gondii and Plasmodium species, although the pathway differs in several aspects from that of the hosts [17].

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Sources and fates of metabolites