**5. Activation of oxygen**

Oxygen is essential for energy metabolism and respiration but is has been implicated in many disease and degenerative conditions (Ceconi *et al*., 2003). Activation of oxygen may occur by two different mechanisms: absorption of sufficient energy to reverse the spin on one of the unpaired electrons and monovalent reduction. Non-activated oxygen is a biradical. It can be activated by either reversing the spin on one of the unpaired electrons to form the singlet state or by reduction. In the monovalent reduction of oxygen, superoxide (O2-), hydrogen peroxide (H2O2), hydroxyl radical (OH) and finally, water (H2O) is formed. Superoxide forms the hydroxyl radical (OOH) which is the protonated form of the superoxide anion radical (Gebick and Bielski, 1981; Ceconi *et al.,* 2003).

Numerous enzymes (peroxidases) use hydrogen peroxide as a substrate in oxidation reactions involving the synthesis of complex organic molecules. Haber and Weiss (1994) identified the hydroxyl radical as the oxidizing species in the reaction between H2O2 and ferrous salts.

$$\text{Fe}^{2+} + \text{H}\_2\text{O}\_2 \quad \longrightarrow \longrightarrow \text{Fe}^{3+} + \text{OH} + \text{OH}^-$$

Most oxygen is consumed by the cytochrome oxidase enzyme in the mitochondrial electron transport system. Isolated mitochondria produce H2O2 and O2- in the presence of NADH (Loschen *et al*., 1974). The various Fe-S-proteins and NADH dehydrogenase have also been implicated as possible sites of superoxide and hydrogen peroxide formation (Waterfall *et al*., 1997). Various oxidative processes including oxidation hydroxylations, dealkylations, deaminations, dehalogenation and desaturation occur in the smooth endoplasmic reticulum. Mixed function oxygenases that contain a heme moiety add an oxygen atom into an organic substrate using NADPH as the electron donor. The generalized reaction catalyzed by cytochrome P450 is:

$$\text{RH} + \text{NADPH} + \text{H}^+ + \text{O}\_2 \longrightarrow \text{ROH} + \text{NADP}^+ + \text{H}\_3\text{O}$$

Superoxide is produced by microsomal NADPH dependent electron transport involving cytochrome P450 (Valko *et al*., 2007). One possible site at which this may occur is shown in Figure 1:

Fig. 1. Schematic presentation of the cytochrome P450 electron transport (Valko *et al.,* 2007).

In the peroxisomes and glyoxysomes, compartmentalized enzymes involved in the Boxidation of fatty acids and glyoxylic acid cycle includes glycolate oxidase, catalase and various peroxidases. Glycolate oxidase produces H2O2 in a two-electron transfer from glycolate to oxygen (Lindqvist *et al*., 1991). Xanthine oxidase, urate oxidase and NADH oxidase generate superoxide as a consequence of the oxidation of their substrates (Fridovich, 1970). The xanthine oxidase reaction is often used *in vitro* as a source of superoxide producing one mole of superoxide during the conversion of xanthine to uric acid (Fridovich, 1970). A superoxide-generating NADPH oxidase activity has been clearly identified in plasmalemmaenriched fractions (Valko *et al*., 2007). These flavopoteins may produce superoxide by the redox cycling of certain quinones or nitrogenous compounds and NADPH oxidase reduces Fe3+ to Fe2+ converting it to a form that can be transported. Dysfunction of NADPH oxidase results in the formation of superoxide (Maxwell & Lip, 1997).

2+ 3+ - Fe + H O Fe + OH + OH 2 2 ⎯⎯→

Most oxygen is consumed by the cytochrome oxidase enzyme in the mitochondrial electron transport system. Isolated mitochondria produce H2O2 and O2- in the presence of NADH (Loschen *et al*., 1974). The various Fe-S-proteins and NADH dehydrogenase have also been implicated as possible sites of superoxide and hydrogen peroxide formation (Waterfall *et al*., 1997). Various oxidative processes including oxidation hydroxylations, dealkylations, deaminations, dehalogenation and desaturation occur in the smooth endoplasmic reticulum. Mixed function oxygenases that contain a heme moiety add an oxygen atom into an organic substrate using NADPH as the electron donor. The generalized reaction catalyzed by

+ + RH + NADPH + H + O ROH + NADP + H O 2 3 ⎯⎯→

Superoxide is produced by microsomal NADPH dependent electron transport involving cytochrome P450 (Valko *et al*., 2007). One possible site at which this may occur is shown in

Fig. 1. Schematic presentation of the cytochrome P450 electron transport (Valko *et al.,* 2007). In the peroxisomes and glyoxysomes, compartmentalized enzymes involved in the Boxidation of fatty acids and glyoxylic acid cycle includes glycolate oxidase, catalase and various peroxidases. Glycolate oxidase produces H2O2 in a two-electron transfer from glycolate to oxygen (Lindqvist *et al*., 1991). Xanthine oxidase, urate oxidase and NADH oxidase generate superoxide as a consequence of the oxidation of their substrates (Fridovich, 1970). The xanthine oxidase reaction is often used *in vitro* as a source of superoxide producing one mole of superoxide during the conversion of xanthine to uric acid (Fridovich, 1970). A superoxide-generating NADPH oxidase activity has been clearly identified in plasmalemmaenriched fractions (Valko *et al*., 2007). These flavopoteins may produce superoxide by the redox cycling of certain quinones or nitrogenous compounds and NADPH oxidase reduces Fe3+ to Fe2+ converting it to a form that can be transported. Dysfunction of NADPH oxidase

results in the formation of superoxide (Maxwell & Lip, 1997).

cytochrome P450 is:

Figure 1:
