Proposed scheme for the electron transport chain in T. gondii

A model for the electron transport chain was recently proposed for Plasmodium (VAN

DOOREN, 2006) (Fig. 4.4). The electron transport chain of T. gondii has most of the components proposed for that of Plasmodium. However, in T. gondii, two isoforms of alternative NADH dehydrogenase (TgNDH2-I and TgNDH2-II ) have been identified instead of one isoform in Plasmodium (PfNDH2). Moreover, the mitochondrion of T.

gondii has a matrix localized NAD-dependent malate dehydrogenase and a membrane bound, FAD-dependent malate:quinone oxidoreductase (FLEIGE, PhD thesis, 2006), while only FAD-dependent malate:quinone oxidoreductase have been identified in Plasmodium (VAN DOOREN et al., 2006)

Based on previous biochemical studies and the bioinformatic analysis of T. gondii genome, we can propose the following possible composition and functions for the electron transport chain in T. gondii.

Electrons can be donated to coenzyme Q from a variety of mitochondrial inner membrane dehydrogenases. As in Plasmodium (Fig. 4.4) five such enzymes exist in T. gondii, although the role of several of these is not yet clearly defined.

Beside the two isoforms of alternative NADH dehydrogenase and succinate dehydrogenase (complex II), the following enzymes are present in the electron transport chain of T. gondii:

(1). Malate is converted to oxaloacetate to complete the tricarboxylic acid cycle, a reaction that is typically catalyzed by NAD-dependent malate dehydrogenase. Instead, the genome of T. gondii reveals as in Plasmodium, the presence of a membrane bound malate-quinone oxidoreductase homologue (TgTwinScan_0081). Malate-malate-quinone oxidoreductase is an enzyme found in some bacteria, and reduces FAD in the generation of oxaloacetate.

Electrons from FADH2 are then donated to coenzyme Q and the electron transport chain.

(2). Dihydroorotate dehydrogenase (DHODH), an enzyme involved in pyrimidine biosynthesis, which catalyzes the oxidation of dihydroorotate to orotate, donating electrons to coenzyme Q via a FAD cofactor. T. gondii dihydroorotate dehydrogenase (TgTwinScan_1012) localizes to the mitochondrion, most probably to the inner membrane (ASAI et al., 1983). In the biosynthesis of pyrimidines, the T. gondii electron transport chain functions as an electron sink.

(3). FAD-linked, or mitochondrial, glycerol-3-phosphate dehydrogenase forms part of the glycerol-3-phosphate shuttle. This shuttle essentially provides an alternative means of

DISCUSSION 112.

directing electrons from cytosolic NADH to mitochondrial coenzyme Q. The first step in this shuttle involves cytosolic NAD⁺-linked glycerol-3-phosphate dehydrogenase, which reduces dihydroxyacetone phosphate (DHAP) to glycerol-3-phosphate, oxidizing cytosolic NADH in the process. Glycerol-3-phosphate is then transported to the mitochondrial intermembrane space, where mitochondrial glycerol-3-phosphate dehydrogenase oxidizes glycerol-3-phosphate back to dihydroxyacetone phosphate, with the electron donated to its FAD cofactor. In turn, this electron is passed into the electron transport chain via coenzyme Q. The T. gondii genome has homologues of both NAD⁺-linked (TgTwinScan_2421) and FAD-linked (TgTwinScan_7069) glycerol-3-phosphate dehydrogenases. However, glycerol 3-phosphate was not able to stimulate ADP phosphorylation in digitonin permeabilized tachyzoites of T. gondii, though it is often taken by the mitochondria (VERCESI et al., 1998).

Figure 4.4: A putative model for electron transport through the inner membrane of the mitochondrion of Plasmodium. Several proteins and protein complexes localize to the inner membrane of mitochondria, where they function in the accepting and donating of electrons.

Several of these enzymes (bottom of diagram) donate electrons to coenzyme Q (CoQ; yellow sphere). Those with clear homologues in T. gondii include succinate dehydrogenase, malate : quinone oxidoreducatase, dihydroorotate dehydrogenase, glycerol-3-phosphate dehydrogenase and NAD(P)H dehydrogenase. It is unclear whether NAD(P)H dehydrogenase oxidizes NAD(P)H derived from the cytosol or from the mitochondrial matrix. Electrons from coenyzme Q are donated to complex III (cytochrome c reductase; top of diagram), which passes electron through to cytochrome c and translocates protons (H⁺) from the matrix into the intermembrane space. Cytochrome c is a soluble intermembrane space protein that donates electrons to complex IV (cytochrome c oxidase), with oxygen functioning as the terminal electron acceptor. Complex IV also translocates protons across the inner membrane. The proton gradient generated by complexes III and IV is harnessed by the F0F1 ATP synthase complex for the production of ATP. (VAN DOORENet al., 2006)

DISCUSSION 113.

In summary, bioinformatic and biochemical evidence suggests that electrons are donated to the Toxoplasma electron transport chain via the FAD-linked tricarboxylic acid cycle enzymes malate : quinone oxidoreductase and succinate dehydrogenase (complex II).

Electrons are also donated during pyrimidine biosynthesis via DHODH. The contribution of electrons donated from cytosolic and mitochondrial NADH and NADPH to the electron transport chain requires further elucidation of the functions and localizations of NAD(P)H dehydrogenase and glycerol-3-phosphate dehydrogenases.

Based on previous biochemical studies and the bioinformatic analysis, we can propose several possible functions for the electron transport chain in T. gondii. It clearly functions as an electron sink for the dihydroorotate dehydrogenase reaction of the essential pyrimidine biosynthesis pathway (LOPEZ et al., 2006). It is clear that the electron transport chain of T. gondii functions in generating a ΔΨ across the inner membrane. It remains to be determined whether ΔΨ has a biologically important role in these parasites. By analogy with other systems, ΔΨ may be required for protein import and in the transport of solutes (LALOI, 1999; PFANNER and GEISSLER, 2001). Interestingly, one study has shown that the addition oligomycin, an inhibitor of mitochondrial ATP synthase, caused an increase in cytosolic Ca²⁺ levels in tachyzoites. This increase suggested a requirement for mitochondrial energy for the regulation of cellular Ca²⁺ homeostasis in these parasites (MORENO and ZHONG, 1996).

The ATP proportion generated by glycolysis and oxidative phosphorylation in T. gondii remains an opened question. However, it is tempting to speculate that in tachyzoite stage most of the ATP is generated by glycolysis, a minimal amount of ATP is generated coupled to electrons donated from pyrimidine biosynthesis, and perhaps by the small amount of NADH generated during glycolysis via the glycerol-3-phosphate shuttle and/or an external NADH dehydrogenase(s).

REFERENCES 114