1.2.1 Biosynthesis of Superoxide
Biological sources of superoxide can originate from all processes, in which oxygen is somehow activated or the redox equilibrium is disturbed [35]:
• The respiratory chain in mitochondria (e.g. cytochrome c oxidase and other heme and heme-thiolate proteins) [35, 36, 37].
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• Monooxygenation and oxygenation reactions [35, 38].
• Conversion of xanthine DH into xanthine oxidase (XO) [35, 39, 40, 41].
• Aggregation of certain protein fragments in membranes exhibits the NADH/NADPH oxidase activity [35, 43, 44].
• Activated macrophages, phagocytosis (NADH/NADPH oxidase activity) [35, 44].
• Autoxidation of polyphenols or fatty acids [35, 45].
• Under certain circumstances heme-thiolate enzymes can be modified and converted to superoxide-producing proteins. NOS has been reported to produce superoxide after treatment with PN [46].
For normal concentrations of superoxide the organism has two protection enzymes: Mn-SOD (only in mitochondria) and Cu,Zn-SOD in the cytosol [47].
Especially XO and NADPH/NADH oxidase seem to be important sources for high concentrations of superoxide. XDH from liver and milk is converted to XO by either thioloxidation and/or proteolysis by which the affinity for oxygen is increased dramatically [39, 41]. The active site of XO consists of two metal centers, a Mo-containing one and an iron-sulfur cluster [48, 41].
Inbetween these two metal centers the binding sites for FAD and NAD+ are localized [40, 41]. The Mo-coordinating sulfurs are part of a Mo-pterin-cofactor. XO is inhibited by tungsten, cyanide and allopurinol, which is oxidized to 2-oxo-allopurinol and reversibly inhibits the active site [39]. XO shows only low substrate specificity and converts hypoxanthine, xanthine, NADH/NADPH (XO has also NADH/NADPH oxidase activity) and even acetaldehyde [22]. Fig.3 shows the major steps in the postulated mechanism of superoxide formation, which was combined from a talk of Prof. Daleand [42].
The phagocyte NADPH oxidase flavocytochrome b558 is a heterodimer and consists of a glycosylated subunit, gp91(phox), and a nonglycosylated one, p22(phox), which after assembling in the presence of two other components (p47phox, p67phox) in the membrane show the O·−2 -generating activity [43, 44].
This process is started after phosphorylation by phosphokinase C (PKC). NADPH oxidase contains two nonidentical heme groups that mediate the final steps of elec-tron transfer. NADPH oxidase activity is stimulated by angiotensin-II [49]. This stimulation could not be observed in cell lines which were deficient in one of the subunits.
Figure 3: Proposed mechanism for the formation of superoxide from XO and xanthine.
The iron-sulfur cluster is also necessary for the full activity of the enzyme, but its role in this mechanism is still not clarified. During the conversion of xanthine to uric acid two molecules of superoxide are formed from oxygen. This mechanism was combined from a talk of Prof.
Daleand [42].
1.2.2 Chemical Sources and Determination of Superoxide
Often a solution of KO2 in DMSO is used as a source for superoxide [50] and to obtain low stationary concentrations of O·−2 the DMSO solution can be added by a constant minimal flow. Another method described for the formation of superoxide is the autoxidation of pyrogallol in air-saturated solutions. The flux of O·−2 formation can be controlled by the pH (in alkaline solutions the autoxidation is faster) and temperature [51]. The latter method is not suitable for spec-troscopic measurements, since the oxidation products of pyrogallol (quinones, semiquinones) themselves show high absorbancies between 250 and 500 nm. The best system to produce constant fluxes of O·−2 in vitro consists of the biological system XO/hypoxanthine in which hypoxanthine is oxidized to xanthine in a first step and to uric acid in a second one and oxygen is reduced to superoxide [22].
Superoxide can be determined by reduction of ferricytochrome c (Fe3+) to ferrocytochrome c (Fe2+) [21]. The reaction is followed at 550 nm. Another possibility for the detection of O·−2 is the oxidation/reduction of fluorescence dyes or chemiluminogenic compounds (e.g. lucigenin). Both methods are not specific for superoxide, therefore one has to take care of other oxidizing and reducing species in the reaction solution.
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1.2.3 Chemistry of Superoxide
At pH 7, superoxide is a shortlived radical with a rather low reactivity. Its short lifetime is due to its fast disproportion in aqueous solutions. By the reaction with metals and other reactive species superoxide can generate hydroxyl radicals, which may damage nearly all existing biomolecules. In vivo there are two enzymatic systems which keep the O·−2 concentration low, the Mn-SOD and Cu,Zn-SOD.
The following equations show the most important reactions of superoxide and its major decomposition product hydrogen peroxide:
• Disproportionation of O·−2 in aqueous solution with k=7.3 x 105 M−1s−1 for the self-dismutation [50], k=108 M−1s−1 for the Mn-SOD catalyzed reac-tion [50], k=2 x 109 M−1s−1 under catalysis of Cu,Zn-SOD [50] and k=107 M−1s−1 in presence of Mn- and Fe-porphyrins [50]. Hydrogen peroxide is scavenged by catalase (k=0.8-2 x 107 M−1s−1)[52]:
2O·−2 + 2H+ −→ O2 + H2O2 (20)
2O·−2 + 2H+ −→SOD O2 + H2O2 (21) 2H2O2 −→Cat O2 + 2H2O (22)
• Reaction with transition metals (Fe(III), Cu(I) and (II), Ni(II) and Co(II)), e.g. Fenton reaction (24) and Haber-Weiss cycle (25)[3, 53, 54]:
Fe3+ + O·−2 −→ Fe2+ + O2 (23)
Fe2+ + H2O2 −→ Fe3+ + ·OH + OH− (24) H2O2 + O·−2 Fe(III)−→ O2 + OH− + ·OH (25)
• Reaction with hypohalogenites (X=Cl−, Br−, I−) [3, 53]:
O·−2 + HOX −→ O2 + ·OH + X− (26)
• Reactions with thiols leads to thiyl radical and disulfide formation [1]:
O·−2 + RSH + H+ −→ H2O2 + RS· −→2x RSSR (27)
• Oxidations by the perhydroxyl radical (pKa=4.5), e.g. lipidperoxidation of polyunsaturated fatty acids(28)and radical chain reaction of polyphenolic compounds (29)[1]:
R−CH = CH−CH2−CH = CH−R + HOO· −→ (28) R−CH = CH−C·H−CH = CH−R + H2O2 →→
A−OH + O·−2 → A−O−+ HOO· → A−O·+HOO− (29)
• The reaction with nitric oxide will be discussed in more detail in 1.3.1.
Superoxide is unlikely to react directly with biomolecules, but in its proto-nated form, as the perhydroxyl radical it is much more reactive and additionally to unsaturated lipids and polyphenols reacts with antioxidants such as ascorbate and tocopherols [1]. Especially in the presence of transition metals superoxide shows a high oxidative potential, mainly mediated by hydroxyl radicals and hy-drogen peroxide [53].
1.2.4 Biology of Superoxide
There are only few indications for physiological actions of superoxide. It is well known that O·−2 is produced in macrophages during phagocytosis [55] and this action can be interpreted as an unspecific immune defense. Furthermore super-oxide could participate in signal transduction, because nearly all cell types show increased levels of O·−2 , when they are stimulated by cytokines. Therefore O·−2 could be a kind of unspecific second messenger. At least superoxide plays a ma-jor role as an intermediate during oxygen activation, but this physiological action should proceed protein-bound and caged. So controlled neither free superoxide should escape the cage nor other reactive species, which are formed during the catalytic cycle of this oxygen activation. The metal-oxo intermediates will be discussed in more detail.