What has been left out of the preceding discussion is the identification of the species responsible for oxidative damage, i.e., the agents that directly attack the various vulnerable targets in the cell. They were left out because the details of "- the chemistry responsible for dioxygen toxicity are largely unknown. In 1954, Rebeca Gerschman formulated the "free-radical theory of oxygen toxicity" after noting that tissues subjected to ionizing radiation resemble those exposed to elevated levels of dioxygen. 35 Fourteen years later, Irwin Fridovich proposed that the free radical responsible for dioxygen toxicity was superoxide, O2 , based on his identification of the first of the superoxide dismutase enzymes.
Today it is still not known if superoxide is the principal agent of dioxygen toxicity, and, if so, what the chemistry responsible for that toxicity is. 6 There is no question that superoxide is formed during the normal course of aerobic metabolism,121 although it is difficult to obtain estimates of the amount under varying conditions, because, even in the absence of a catalyst, superoxide disproportionates quite rapidly to dioxygen and hydrogen peroxide (Reaction 5.4) and therefore never accumulates to any great extent in the cell under normal conditions of pH. 37
One major problem in this area is that a satisfactory chemical explanation for the purported toxicity of superoxide has never been found, despite much indirect evidence from in vitro experiments that the presence of superoxide can lead to undesirable oxidation of various cell components and that such oxidation can be inhibited by superoxide dismutase. 38 The mechanism most commonly proposed is production of hydroxyl radicals via Reactions (5.28) to (5.30) with Red - = O2-, which is referred to as the "Metal-Catalyzed Haber-Weiss Reaction". The role of superoxide in this mechanism is to reduce oxidized metal ions, such as Cu2 + or Fe 3+ , present in the cell in trace amounts, to a lower oxidation stateY Hydroxyl radical is an extremely powerful and indiscriminate oxidant. It can abstract hydrogen atoms from organic substrates, and oxidize most reducing agents very rapidly. It is also a very effective initiator of freeradical autoxidation reactions (see Section II.C above). Therefore, reactions that produce hydroxyl radical in a living cell will probably be very deleterious. 6
The problem with this explanation for superoxide toxicity is that the only role played by superoxide here is that of a reducing agent of trace metal ions. The interior of a cell is a highly reducing environment, however, and other reducing agents naturally present in the cell such as, for example, ascorbate anion can also act as Red - in Reaction (5.28), and the resulting oxidation reactions due to hydroxyl radical are therefore no longer inhibitable by SOD.39 Other possible explanations for superoxide toxicity exist, of course, but none has ever been demonstrated experimentally. Superoxide might bind to a specific enzyme and inhibit it, much as cytochrome oxidase is inhibited by cyanide or hemoglobin by carbon monoxide. Certain enzymes may be extraordinarily sensitive to direct oxidation by superoxide, as has been suggested for the enzyme aconitase, an iron-sulfur enzyme that contains an exposed iron atom. 122 Another possibility is that the protonated and therefore neutral form of superoxide, H02 , dissolves in membranes and acts as an initiator of lipid peroxidation. It has also been suggested that superoxide may react with nitric oxide, NO, in the cell producing peroxynitrite, a very potent oxidant. 123 One particularly appealing mechanism for superoxide toxicity that has gained favor in recent years is the "Site-Specific Haber-Weiss Mechanism." 40,41 The idea here is that traces of redox-active metal ions such as copper and iron are bound to macromolecules under normal conditions in the cell. Most reducing agents in the cell are too bulky to come into close proximity to these sequestered metal ions. Superoxide, however, in addition to being an excellent reducing agent, is very small, and could penetrate to these metal ions and reduce them. The reduced metal ions could then react with hydrogen peroxide, generating hydroxyl radical, which would immediately attack at a site near the location of the bound metal ion. This mechanism is very similar to that of the metal complexes that cause DNA cleavage; by reacting with hydrogen peroxide while bound to DNA, they generate powerful oxidants that react with DNA with high efficiency because of their proximity to it (see Chapter 8).
The problem with this explanation for superoxide toxicity is that the only role played by superoxide here is that of a reducing agent of trace metal ions. The interior of a cell is a highly reducing environment, however, and other reducing agents naturally present in the cell such as, for example, ascorbate anion can also act as Red - in Reaction (5.28), and the resulting oxidation reactions due to hydroxyl radical are therefore no longer inhibitable by SOD.39 Other possible explanations for superoxide toxicity exist, of course, but none has ever been demonstrated experimentally. Superoxide might bind to a specific enzyme and inhibit it, much as cytochrome oxidase is inhibited by cyanide or hemoglobin by carbon monoxide. Certain enzymes may be extraordinarily sensitive to direct oxidation by superoxide, as has been suggested for the enzyme aconitase, an iron-sulfur enzyme that contains an exposed iron atom. 122 Another possibility is that the protonated and therefore neutral form of superoxide, H02 , dissolves in membranes and acts as an initiator of lipid peroxidation. It has also been suggested that superoxide may react with nitric oxide, NO, in the cell producing peroxynitrite, a very potent oxidant. 123 One particularly appealing mechanism for superoxide toxicity that has gained favor in recent years is the "Site-Specific Haber-Weiss Mechanism." 40,41 The idea here is that traces of redox-active metal ions such as copper and iron are bound to macromolecules under normal conditions in the cell. Most reducing agents in the cell are too bulky to come into close proximity to these sequestered metal ions. Superoxide, however, in addition to being an excellent reducing agent, is very small, and could penetrate to these metal ions and reduce them. The reduced metal ions could then react with hydrogen peroxide, generating hydroxyl radical, which would immediately attack at a site near the location of the bound metal ion. This mechanism is very similar to that of the metal complexes that cause DNA cleavage; by reacting with hydrogen peroxide while bound to DNA, they generate powerful oxidants that react with DNA with high efficiency because of their proximity to it (see Chapter 8).
Although we are unsure what specific chemical reactions superoxide might undergo inside of the cell, there nevertheless does exist strong evidence that the superoxide dismutases play an important role in protection against dioxygeninduced damage. Mutant strains of bacteria and yeast that lack superoxide dismutases are killed by elevated concentrations of dioxygen that have no effect on the wild-type cells. This extreme sensitivity to dioxygen is alleviated when the gene coding for a superoxide dismutase is reinserted into the cell, even if the new SOD is of another type and from a different organism.
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