Cryptosporidium is a protist causing serious diarrhea in hum
Cryptosporidium is a protist causing serious diarrhea in humans and other animals (Bouzid et al. 2013). In its mitosomes, the most reduced forms of mitochondria, it is assumed that the membrane potential is generated by a simple respiratory chain consisting of transhydrogenase, type II NADH dehydrogenase, and AOX (Mogi and Kita 2010). In some Cryptosporidium, a unique respiratory chain consisting of externally bound NDH2 and internally bound AOX can reoxidize cytoplasmic NADH. ATP and NADPH are produced in the mitosome and could be used for iron-sulfur cluster formation. The genome of Toxoplasma gondii, the parasite that causes toxoplasmosis, encodes two NDH2 isoforms and both are constitutively expressed (TgNDH2-I and TgNDH2-II) (Lin et al. 2008). The two TgNDH2 isoforms are internal enzymes that face the mitochondrial matrix with their active sites. Knockout experiments indicated that both Tgndh2 genes are required to maintain normal mitochondrial membrane potential and the intracellular ATP level, although TgNDH2-II appears to be more important (Lin et al., 2011). TgNDH2-I has been heterologously expressed in Y. lipolytica mitochondria and its kinetic parameters have been determined (Lin et al. 2008). Kinetic studies have revealed that the NADH dehydrogenase activity of TgNDH2-I follows a ping-pong mechanism, a mode of action also shown for the Y. lipolytica ortholog and proposed for the S. cerevisiae and T. brucei enzymes. For TgNDH2-I, the KM determined for NADH is equal to 76μM and is significantly higher than the known KM values of most other eukaryotic enzymes. For example, they range from 9–56μM in N. crassa (Velazquez and Pardo 2001) and S. cerevisiae (Duarte et al., 2003, Melo et al., 2004). The exception is the T. brucei enzyme with a KM of ∼120μM (Fang and Beattie 2002a). However, differences in KM values have to be interpreted with caution, since different Metformin acceptors were used during the measurements. TgNDH2-I is inhibited effectively (in nanomolar concentration) by the quinolone-like compound, 1-hydroxy-2-dodecyl-4(1)quinolone (HDQ) and other quinolones possessing longer alkyl side chains. However, it cannot be ruled out whether HDQ inhibits other ubiquinone-dependent oxidoreductases, such as succinate dehydrogenase or glycerol-3-phosphate dehydrogenase, in addition to the alternative NADH dehydrogenases (Fang and Beattie 2002a). AmoebaThe amoeba Acanthamoeba castellanii is a small, nonphotosynthesizing free-living soil and freshwater protozoan that can cause serious diseases in humans, such as Acanthamoeba keratitis (AK) and granulomatous amoebic encephalitis (GAE) (Marciano-Cabral and Cabral, 2003, Schuster and Visvesvara, 2004). The mitochondria of A. castellanii possess a branched plant type respiratory chain with AOX and alternative NAD(P)H dehydrogenases (Antos-Krzeminska and Jarmuszkiewicz, 2014a, Antos-Krzeminska and Jarmuszkiewicz, 2014b). Annotated sequences of two putative alternative NAD(P)H dehydrogenases from the protozoan A. castellanii demonstrate similarity to plant and fungal sequences and reveal EF-hand motifs indicating Ca2+-binding domains (Antos-Krzeminska and Jarmuszkiewicz 2014a). BN-PAGE electrophoresis and histochemical staining of A. castellanii mitochondrial proteins reveal six protein bands of relatively low molecular mass (∼50–70kDa): three with NADH-oxidizing activity, and the other three with NADPH-oxidizing activity. In isolated A. castellanii mitochondria, external NADPH oxidation has been observed for the first time in protist mitochondria. In A. castellanii mitochondria, external NADH and NADPH oxidation exhibit similar coupling parameters, indicating similar efficiencies of ATP synthesis, and the same optimal pH (6.8) independent of relevant ubiquinol-oxidizing pathways, the cytochrome pathway or a GMP-stimulated AOX-sustained pathway. However, a twice lower maximal oxidizing activity, 10-fold higher Ca2+-dependence, and a lower KM value for external NADPH oxidation were observed compared to those for external NADH oxidation. The results indicate a higher dehydrogenase affinity for external NADPH, and therefore a finely tuned strong Ca2+-dependent regulation of external NADPH oxidation in the A. castellanii mitochondria. Remarkably, external NADH is in addition to succinate (Complex II substrate) one of the strongest respiratory substrates, indicating a substantial contribution of external NADH dehydrogenase activity to the electron transport chain in the A. castellanii mitochondria and increased substrate (external NADH) availability in the cytosol (Antos-Krzeminska and Jarmuszkiewicz 2014a).