The Pathway of Electron Transport: The respiratory Chain and superoxide formation

 The concept that a chain of electron carriers is responsible for transferring electrons from substrate molecules to molecular oxygen represents the confluence of two lines of investigations. Early investigators of biological oxidations in the period 1900-1920, particularly T. Thunberg, had discovered the dehydrogenases, which catalyze removal of hydrogen atoms from different metabolites in the complete absence of oxygen. From such experiments H. Wieland later postulated that activation of hydrogen atoms is the basic process involved in biological oxidation and that molecular oxygen does not need to be activated to react with the active hydrogen atoms yielded by dehydrogenases. However, in 1913 O. Warburg discovered that cyanide in very small concentrations almost completely inhibits the oxygen consumption of respiring cells and tissues. Since, cyanide does not inhibit dehydrogenases but does form very stable complexes with iron, Warburg postulated that biological oxidation requires an iron containing enzyme; in his view activation of oxygen by this enzyme is the basic mechanism involved in biological oxidation. The differing views of Wieland and Warburg, i.e., hydrogen activation vs. oxygen activation, were later brought together by A. Szent-Gyorgyi in Hungary, who postulated that both processes take place and that flavoproteins play the role of intermediate electron carriers between the dehydrogenases and the respiratory enzyme. Keilin also provided important evidence that the cytochrome act as a consecutive series of electron carriers. He and other investigators, among them D.E. Green, K.Okunuki, T. Singer, T. King, and E. Racker, carried out in vitro reconstructions of segments of the electron-transport chain starting from purified components.

            The sequence of electron transfer reactions in the respiratory chain from NADH to oxygen is now fairly well established. NADH is the form in which electrons are collected from many different substrates through the action of NAD-linked dehydrogenases. These electrons funnel into the chain via the flavoprotein NADH dehydrogenase. On the other hand, other respiratory substrates are dehydrogenated by flavin-linked dehydrogenase, such as succinate dehydrogenase and acetyl-CoA dehydrogenase, which funnel electrons into the chain via ubiquinone. NAD⁺ and ubiquinone thus serve to collect by pyridine-linked and flavin-linked dehydrogenases, respectively, again illustrating the principle of convergence in catabolic pathway.

          The sequence from NAD to oxygen is supported by several lines of evidence. First, it is consistent with the standard oxidation-reduction potentials of the different electron carriers, which become more positive as electrons pass from substrate to oxygen. Second, in vitro reconstruction experiments with isolated electron carriers have shown that NADH can reduce NADH dehydrogenase but cannot directly reduce cytochrome b, c, or cytochrome aa_3. Similarly, reduced NADH dehydrogenase cannot react directly with cytochrome b and c₁. Third, complexes containing groups of functionally linked carriers have been isolated from mitochondria, e.g., a complex of cytochromes b and c₁ and an iron –sulfur protein, and a complex of NADH dehydrogenase and one or more iron-sulfur proteins.

            Perhaps the most important and direct evidence has come from measurements of the oxidation-reduction state of the individual electron carriers as they function in intact mitochondria, by measurement of difference spectra, a procedure developed by B. Chance and G.R. Williams. Because mitochondria suspensions are turbid and absorb and scatter much light, the absorption spectra of the electron carriers in intact mitochondria cannot be measured by direct spectrophotometry. However, it is possible to measure the amounts of the carriers in their reduced states in such a turbid suspension by reading its optical absorption in a sensitive spectrophotometer against a blank or control suspension of mitochondria in which the carriers are in their oxidized state, thus canceling out the large absorption due to the turbidity of the suspension. When isolated mitochondria in the aerobic steady state are allowed to oxidize intermediates of the tricarboxylic acid cycle, difference spectra show that the electron carrier nearest the reducing or substrate end of the chain in the aerobic steady state whereas the electron carriers at the oxygen end is almost entirely in the oxidized form. The intermediate electron carriers of the chain, in the direction from substrate to oxygen, are present in successively more oxidized form in the aerobic steady-state, indicating that the electrons flow along a gradient from NAD to oxygen. In another spectrophotometric approach measurements have been made of the rate and sequence of re-oxidation of the carriers when oxygen is admitted to an anaerobic suspension of mitochondria in the presence of excess substrate. Under anaerobic conditions all the carriers are fully reduced; when oxygen is admitted, reduced cytochrome aa₃ becomes oxidized first, followed by cytochrome c, and then cytochrome b, which is in turn followed by re-oxidation of NADH. It is important to emphasize that a difference spectrum is not the spectrum of the reduced form of the electron carriers, but the difference between the spectra of the reduced and oxidized forms.

Some Uncertainties in the Electron-Transport Chain of Mitochondria

Although much evidence indicates that iron-sulfur proteins or centers are involved at several points in the chain of electron carriers from NADH to oxygen, their precise location and function are not yet known. It may also be pointed out that cytochrome b is now known to occur in two forms, cytochrome b_k and cytochrome b_t, differing in their standard oxidation-reduction potentials. The function of these two forms is not yet clear, although cytochrome b_T has been postulated to function in the mechanism of energy transduction during electron transport.

            Uncertainity involves, the number of electrons transferred in each step of the respiratory chain. It is generally believed that electron transport occurs in two-electron steps between NAD and ubiquinone and in one-electron steps from cytochrome b to oxygen. On the other hand, the reduction of one molecule of oxygen to two water molecules requires a total of four electrons. How electron flow in the respiratory chain is coordinated to yield four electrons to ensure complete reduction of an O₂ molecule is not yet known. One suggestion is that the cytochromes may function in pairs. This is an especially important question since partial reduction of the oxygen molecule yields extremely toxic products. Reduction by a single electron yields the superoxide radical O₂^-, whereas reduction by two electrons yields hydrogen peroxide. Both products are highly reactive and potentially destructive to certain types of functional groups present in biomolecules. There is now increasing evidence that superoxide and hydrogen peroxide are in fact produced during reduction of oxygen in animal tissues. 

Superoxide Dismutase and Catalase

During electron transport to molecular oxygen via the mitochondrial respiratory chain, as well as in various hydroxylation and oxygenation reactions, toxic partial reduction products of oxygen may be formed, presumably as transient intermediates on the active sites of such enzymes. The most important are the superoxide anion O₂^- and hydrogen peroxide which are extremely reactive and capable of irreversible damage to various biomolecules.

            It has been found, largely through the research of I. Fridovich and his colleagues, that aerobic cells generally contain the enzyme superoxide dismutase, which converts superoxide into hydrogen peroxide and molecular oxygen. Superoxide dismutase is found in two forms, one in the extramitochondrial cytosol and another in the mitochondria. The mitochondrial superoxide dismutase of eukaryotes is similar to the superoxide dismutase of many bacteria with respect to its characteristic content of Mn²⁺ and many homologies in amino acid sequence. The cytosol form of superoxide dismutase, on the other hand, has quite a different structure and contains Cu²⁺ and Zn²⁺. These enzymes are present in high concentration and are extraordinary active, suggesting those superoxide radicals are being continuously produced during the enzymatic reduction of oxygen by various enzymes and enzyme systems and quickly removed. Hydrogen peroxide formed by superoxide dismutase and by the flavin-linked oxidases is decomposed by the heme enzyme catalase in the reaction. Catalase is found in the microbodies of animal cells, also called peroxisomes. The protective action of superoxide dismutase and catalase is probably supported by ascorbic acid, glutathione and vitamin E, which readily accept electrons and may serve a backup function by scavenging free radicals.

Source:-Biochemistry Second Edition, The Molecular Basis of Cell Structure and Function. Albert L. Lehninger, The Johns Hopkins University School of Medicine.