As indicated already, membrane-associated electron transport has not been demonstrated in mitosomes. Neither cytochromes nor haem proteins have been detected by chemical or spectroscopic analyses in anaerobic protozoa and no components of respiratory complexes I-IV have been identified in the genomes of C. parvum, E. cuniculi, E. histolytica and G. intestinalis (Abrahamsen et al. 2004; Katinka et al. 2001; Loftus et al. 2005; McArthur et al. 2000; Müller 2003). However, low levels of ubiquinone have been detected in Giardia and Entamoeba (Ellis et al. 1994). A limited level of membrane-associated electron transport activity in C. parvum is suggested by the presence of a gene encoding alternative oxidase (AO) in its genome. This enzyme transfers electrons to molecular oxygen and its catalytic activity is associated with thioredoxin reductase and glutathione peroxidase in plants and fungi (Abrahamsen et al. 2004; Putignani et al. 2004). Because these two enzymes are also present in the C. parvum genome, a previously unidentified oxygen-scavenging mechanism might operate in this organism; its functionality and the precise cellular localisation of AO remain to be determined experimentally.
The observed reduction of 5-cyano-2,3,-ditolyl tetrazolium chloride (CTC) and concomitant formazan precipitation in Giardia was recently taken as evidence for the presence of mitochondrial electron transport in this organism (Lloyd et al. 2002a). However, the specificity of tetrazolium salt reduction in Giardia is questionable as tetrazolium salt reducing systems have been demonstrated in the internal membranes and in the plasma membrane of mammalian cells (Bernas and Dobrucki 2000). Co-localisation experiments with organelle-specific molecular markers are required to ascertain the nature of CTC reduction in Giardia. Uptake of rhodamine 123, another fluorescent cation, has also been used in Giardia and Blastocystis as evidence for the presence of membrane potential in mitosomes (Lloyd et al. 2002a; Nasirudeen and Tan 2004; Zierdt et al. 1988). Staining of Blastocystis mito-somes is inhibited by the addition of sodium azide, which affects the membrane potential through inhibition of cytochrome c oxidase, an activity previously undetected in this organism (Nasirudeen and Tan 2004; Zierdt et al. 1988). As with CTC reduction, the specificity of rhodamine 123 staining in Giardia remains to be validated by co-localisation experiments using organelle-specific molecular markers.
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