8.3Oxidation-Reduction Reactions and the Respiration Chain
In terms of the utilization of biological energy, the ability to convert energies generated by various oxidation-reduction reactions to a single form, H+ electrochemical potential, is a significant advantage. Oxidation-reduction reactions involve the transfer of electrons between two materials, and are mediated mainly by cofactors (i.e., coenzymes). Since the same cofactors (e.g., NAD+) tend to be used in different enzymatic oxidation-reduction reactions in cells, energy can be efficiently produced through a common mechanism by converging enzymatic oxidation-reduction reactions into the respiration chain through the mediation of cofactors. Among the common cofactors, NADH and FADH2 are often utilized, and quinones (such as ubiquinone) and cytochrome c are also used (Fig. 8-1). Most of these cofactors are common to all organisms, and photosynthesis (discussed in 8.5 in this chapter) also uses very similar cofactors. The free energy change of oxidation-reduction reactions has a linear relationship with the difference in oxidation-reduction potential between two reactants, A and B (Equation 8-3).
ΔG˚’ = -nF(E˚’A - E˚’B)
where n is the number of electrons transferred and F is the Faraday constant.
The hydrogen (E˚’ = -0.315 V) in NADH releases 218 kJmol-1 of energy by reacting directly with oxygen (E˚’ = +0.815 V)*4, but the respiration chain is separated into electrons and H+. These electrons, which have a high reducing ability (i.e., low oxidation-reduction potential), gradually release energy via around 20 types of electron carrier and finally react with oxygen in mild conditions. Among these electron carriers, ubiquinone (a low molecular weight compound) (Fig. 8-1) and cytochrome c (a small protein) act as mobile electron carriers, and the rest are contained in four types of protein complex (Complexes I - IV) as cofactors. These cofactors, which strongly bind to proteins, are known as the prosthetic group. In terms of substrate oxidation and reduction, these protein complexes are also called NADH dehydrogenase (Complex I), succinate dehydrogenase (Complex II), cytochrome bc1 complex (Complex III) (also known as ubiquinone-cytochrome c oxidoreductase) and cytochrome c oxidase (Complex IV) (Fig. 8-2*5).
Fig. 8-2. A mitochondrion (top), and the respiration chain and ATP synthase - a protein complex - found in the inner membrane of mitochondria (bottom)
Q (an electron transport component) is a ubiquinone, c, c1, b, a and a3 are hemes (cytochrome cofactors), and FeS is an iron-sulfur cluster cofactor. FMD is flavin mononucleotide, and FAD is flavin adenine dinucleotide. Reduced Q generated by Complex II also passes electrons on to Complex III via the quinone cycle. Since the mechanism of H+ transport is not fully understood, the number of H+ ions transported by each complex is set as n. Overall, approximately three molecules of ATP are synthesized through the complete oxidation of one NADH molecule, and the value of n is approximately 9 in this case.
The free energy change during the complete oxidation of NADH (ΔG˚’= -218 kJmol-1) is equivalent to that of around seven ATP molecules (ΔG˚’ = -30.5 kJmol-1 per molecule); however, only three ATP molecules are actually generated, and the remaining free energy change is used to tilt the equilibrium toward ATP synthesis. The standard reduction potential of other major materials are E˚’ = +0.031 V for succinic acid, E˚’ = +0.045 V for ubiquinone and E˚’ = + 0.235 V for cytochrome c. In other words, the free energy change in the reaction that produces ubiquinone from succinic acid catalyzed by Complex II (succinate dehydrogenase) is insufficient for ATP synthesis, whereas it is large in the enzymatic reactions of Complex I, III and IV, thus allowing ATP synthesis. The inability of the Complex II reaction to generate enough energy to synthesize ATP is consistent with the fact that in the citric acid cycle, FADH2 is generated only at the stage of succinate dehydrogenase and the number of ATP molecules generated by the oxidation of FADH2 is one fewer than in the oxidation of NADH. It can be said that the reducing power of succinic acid is weaker than that of NADH. The number of ATP molecules generated per glucose molecule is therefore 2 in glycolysis, 2 in the citric acid cycle, 4 in the oxidation of 2 FADH2 molecules, and 30 in the oxidation of 10 NADH molecules, making a total of 38 molecules*6.
In the aerobic respiration of mitochondria, glucose can be used for energy production in addition to the NADH and FADH2 generated by the degradation of amino acids and fatty acids (Fig. 8-3). Fatty acids undergo dehydrogenation by two-carbon-atom units (β-oxidation) and enter the citric acid cycle as acetyl-CoA, where they are completely oxidized. Amino groups that constitute amino acids are converted to urea, and the remaining carbon skeletons are completely oxidized in glycolysis and the citric acid cycle for use in ATP production. The mitochondrial respiration chain is thus a key component of energy production.
The main role of the respiration chain is to efficiently transport H+ across the inner membrane to the outside by coupling with the stepwise oxidation and reduction of electron carriers, and the details of the mechanism, including the quinone cycle proposed by Mitchell, have been gradually revealed. Quinones, such as ubiquinone, are lipophilic materials that transfer H+ through oxidation and reduction (Fig. 8-1), and are therefore widely used by various organisms in reactions that create high-energy states by transporting H+ across the membrane. The quinone cycle is a system that transports two H+ ions by the transfer of one electron when electrons are transferred from a ubiquinone to a cytochrome bc1 complex. Such complexes have separate sites for quinone oxidation and reduction, and a reduced ubiquinone bound to an oxidation site releases two H+ ions and two electrons. Of these, one electron is transferred to cytochrome c1 via iron-sulfur clusters, and the other reduces another ubiquinone at the quinone reduction site via cytochrome b. Both H+ ions are subsequently released to the outside of the inner membrane. With the progress of spectroscopy and structural biology in recent years, the structure of these protein complexes and the behavior of each electron carrier have gradually been clarified, and Japan leads the world in the field of research into cytochrome c oxidase.
Calculation example: To obtain the standard free energy change of the two-electron reaction NADH + H+ + 1/2 O2 ⇄ NAD+ + H2O, with O2 (E˚’ = +0.815 V) and NAD (E˚’= -0.315 V),
n = 2 is assigned to Equation 8-3: ΔG˚’ = -2 x 96.5 x [0.815 - (-0.315)] = -218 kJ mol-1
where the Faraday constant is 96.5 kJV-1 mol-1
NADH is a water-soluble coenzyme that is shared by many enzymes. FADH2, on the other hand, binds to certain enzymes, and is a coenzyme (or cofactor) that facilitates the transfer of electrons in enzymatic reactions. Therefore, in Figure. 8-2, NADH is in the matrix, and FADH2 (FAD in the figure - the oxidized form that receives electrons) binds to the inside of Complex II.
38 molecules: This number is for bacteria; the number for mitochondrial ATP synthesis is in fact 36. This is because energy equivalent to that released by two ATP molecules is used to transport two molecules of NADH generated in glycolysis from the cytoplasm to mitochondria.