**2.5 Mitochondrial ROS production and antioxidant enzymes**

Mitochondria are the major source of ROS generation [9]. In an organism, mitochondria utilize approximately 98% of the total amount of inhaled O2, including 1–2% for ROS generation [59, 60]. Mitochondria actually produce ROS in a number of enzymatic reactions; the vast majority of the free radicals from the mitochondria are formed in the ETC during OXPHOS [61]. In the process of OXPHOS, electron leaks from the ETC combine with O2 molecules to form (O2 ·<sup>−</sup>). Mitochondrial O2 ·<sup>−</sup>, primarily generated in complexes I and III, is catalysed by Cu/ZnSOD or Mn SOD to disproportionate into H2O2. Subsequently, H2O2 can be converted to OH· by Fenton reaction. Mitochondrial O2 ·<sup>−</sup> can also bind with protons to form uncharged HOO· radicals and subsequently react with unsaturated fatty acid of mitochondrial membrane lipids to produce lipid radicals. Mitochondrial nitric oxide (NO) interacts with O2 ·<sup>−</sup> to form reactive nitrogen oxide species (RNS) such as peroxynitrite (ONOO<sup>−</sup>), which produce cellular dysfunction by S-nitrosylating proteins [62]. Mammalian cells have multiple enzymes to degrade H2O2, including peroxiredoxins (Prxs), glutathione peroxidases (Gpxs), thioredoxins (Trxs) and catalase. Mitochondrial H2O2 is primarily eliminated by the action of Gpx1, Gpx2 and Gpx4, Prx3 and Prx5 and Trx2 systems, which requires glutathione (GSH) [63–65]. Oxidized GSH (GSSG) is reduced to GSH by glutathione reductase (GR) activity [66]. Similarly, oxidized Trx2 is recycled by Trx reductase (TrxR). These H2O2 scavenging system ultimately depends on reduced nicotinamide adenine dinucleotide phosphate (NADPH) which is regenerated by three mitochondrial matrix-located enzymes: NADP<sup>+</sup> -linked isocitrate dehydrogenase (IDH), malate dehydrogenase (MDH)

#### **Figure 2.**

*Reactions and transformations of mitochondrial ROS. SOD enzymes catalyse the dismutation of superoxide (O2 ·<sup>−</sup>), generating hydrogen peroxide (H2O2). The catalase (CAT), glutathione peroxidases (Gpxs) and peroxiredoxins (Prxs) convert H2O2 into water. H2O2 reacts with redox-active iron to generate the hydroxyl radical (OH· ) through the Fenton reaction. The reaction between O2 ·<sup>−</sup> and nitric oxide (NO· ) produces peroxynitrite (ONOO<sup>−</sup>), whose decomposition in turn gives rise to some highly oxidizing intermediates including NO2 · , OH· , CO3 · as well as, finally, stable NO3 <sup>−</sup>. Thus, increased O2 ·<sup>−</sup> levels can also reduce NO· and generate ONOO<sup>−</sup> toxicity. O2 ·<sup>−</sup> by itself can reduce ferric iron (Fe3+) to ferrous iron (Fe2+) in iron-sulphur centres of proteins, leading to enzyme inactivation and concomitant loss of Fe2+ from the enzymes. The protonation of O2 ·<sup>−</sup> can form the more reactive hydroperoxyl radical (HO2 · ).*

and nicotinamide nucleotide transhydrogenase (NNT) [61]. Catalase catalyses the decomposition of hydrogen peroxide to water and oxygen, existing as a tetramer composed of four identical monomers, each of which contains a heme group at the active site. Catalase also requires NADPH as a reducing equivalent to prevent oxidative inactivation of the enzyme [67] (**Figure 2**).
