*4.1. ROS Production from Mitochondrial Electron Transport Chain*

The mitochondrion constitutes a very important source of free radicals in aerobic organisms. Approximately 1–3% of electrons escape from the electron transport chain directly to react with molecular oxygen, leading to the formation of superoxide radicals (O2 •−) [15]. This is all the more important as the mitochondrion of the parasite responsible for malaria has cytochromes with heme as a cofactor in the electron transport chain [51]. Although there is a paucity of data on the involvement of the parasitic mitochondrion in ROS generation, the complexity and diversity of its antioxidant system is a very good pointer, especially the report of the expression of cytosolic PfSOD-1 and mitochondrial PfSOD-2 throughout the blood stages of the parasite [52]. Superoxide dismutases (SODs) quickly dismutate the formed superoxide radical (O2 •−) to hydrogen peroxide (H2O2), which can be reduced to water by the peroxiredoxin 2-Cys Prx (TPx-2) [53,54].

#### *4.2. ROS Production from Hemoglobin Digestion*

During the blood stage, parasites take up and break down approximately 75% of the hemoglobin (Hb) of red blood cells to obtain essential amino acids for their development and replication, releasing free heme as residual toxic waste [44,55]. This free heme contains reactive ferrous iron (Fe2+) that can readily reoxidize by transferring electrons to oxygen to form superoxide radicals (O2 •−) [16] and then hydrogen peroxide, finally leading to the production of the highly deleterious hydroxyl radical (•OH) through the Fenton reaction involving a new molecule of heme (Fe2+) (Figure 6) [3].

Because of the important role that heme plays in ROS generation, immediately after its production, a detoxification process takes place where approximately 95% of the produced heme (Fe2+) is polymerized into hemozoin, a nontoxic crystal [46]. This reaction involves the heme detoxification protein (HDP), whose action is aided by histidine-rich protein-2 (HRP2) (Figure 7) [48,56]. HRP2 is said to have a very high affinity for heme [57,58], and elevated HRP2 reduces the vulnerability of the parasite. Kapishnikov et al. showed that mature parasites have approximately 70% of the total iron from red blood cells in the hemozoin crystals and therefore suggested a coupling of the rate of Hb digestion to that of heme polymerization [59].

Although *Plasmodium* lacks the heme oxygenase enzyme, which is deployed by most organisms to degrade heme [60], it has a system in place that mimics the heme oxygenase [44]. During the digestion of hemoglobin, H2O2 is also generated in the food vacuole

as a result of the immediate conversion of oxyhemoglobin to methemoglobin due to heme reduction at the prevailing pH of 5.2 [61,62]. The heme is peroxidatively degraded by the reaction with H2O2, leading to the formation of a ferryl intermediate (Fe(IV)=O) [44].

$$\begin{array}{cccc} \text{Fe}^{3+} + \text{O}\_{2} & \underset{\text{Fe}^{2+} + \text{H}\_{2}\text{O}\_{2}}{\text{Fe}^{2+} + \text{H}\_{2}\text{O}\_{2}} & \underset{\text{Fe}^{3+} + \text{OH} + \text{OH}^{\cdot}}{\text{Fe}^{3+} + \text{H}\_{2}\text{O}\_{2}} & \text{(Fenton reaction)}\\ \text{netwodation:} & \text{O}\_{2}\text{-}^{\cdot} + \text{H}\_{2}\text{O}\_{2} & \underset{\text{catalyst}}{\text{Fe}}^{\text{Fe}} & \text{O}\_{2}\text{+}^{\cdot}\text{OH} + \text{OH}^{\cdot}} & \text{(Haber-Weiss reaction)} \end{array}$$

**Figure 6.** Heme Fe2+ reaction with O2 •− leading to ROS production, especially the most deleterious one, the hydroxyl radical •OH [3,63].

**Figure 7.** Hemoglobin digestion and hemozoin production in *Plasmodium*-infected red blood cells. HDP, heme detoxification protein [59,64].

*Plasmodium* uses a family of hemoglobinases to degrade hemoglobin into amino acids. *Plasmodium* also requires heme from the host to synthesize proteins such as the hemedependent cytochromes in the mitochondrial electron transport chain [51], although genetic studies have revealed that the parasites encode and express all the enzymes for heme production [60]. Moreover, the digestion of hemoglobin ensures that the infected red blood cells are osmotically stable throughout the intraerythrocytic stages of the parasite [65]. The digestion of hemoglobin is therefore essential for *Plasmodium* during its intraerythrocytic cycle. In addition to the formation of hemozoin, there are other antioxidant defense mechanisms (discussed below) that can address the toxic effect of resting unpolymerized heme. The significant role played by hemoglobin in oxygen transport makes red blood cells and their vicinity highly vulnerable to ROS formation [66].

**Box 1.** The role of nitric oxide in potentiation of reactive species [15,67,68].
