The second part of the citric acid cycle consists of the regeneration of the starting material, oxaloacetate. This regeneration is accomplished by a series of reactions that begins with a four-carbon molecule and ends with a four-carbon molecule. However, the rearrangements within this set of reactions harvest energy in the form of high-energy electron carriers and a molecule of ATP.

The succinyl CoA produced by α-ketoglutarate dehydrogenase in the preceding step is an energy-rich thioester compound. The ΔG°′ for the hydrolysis of succinyl CoA is about −33.5 kJ mol⁻¹ (−8.0 kcal mol⁻¹), which is comparable to that of ATP (−30.5 kJ mol⁻¹, or −7.3 kcal mol⁻¹). How is this energy utilized in the citric acid cycle? Recall that, in the citrate synthase reaction, the cleavage of the thioester powers the synthesis of the six-carbon citrate from the four-carbon oxaloacetate and the two-carbon fragment. In this case, the cleavage of the thioester of succinyl CoA is coupled to the phosphorylation of a purine nucleoside diphosphate, usually ADP. This reaction is catalyzed by succinyl CoA synthetase (succinate thiokinase):

This reaction is the only step in the citric acid cycle that directly yields a compound with high phosphoryl-transfer potential. In mammals, there are two forms of the enzyme, one specific for ADP and the other for GDP. In tissues that perform large amounts of cellular respiration, such as skeletal and heart muscle, the ADP-requiring enzyme predominates. In tissues that perform many anabolic reactions, such as the liver, the GDP-requiring enzyme is common. The GDP-requiring enzyme is believed to work in reverse of the direction observed in the citric acid cycle; that is, GTP is used to power the synthesis of succinyl CoA, which is a precursor for heme synthesis. The E. coli enzyme uses either GDP or ADP as the phosphoryl-group acceptor.

The mechanism of this reaction is a clear example of an energy transformation: energy inherent in the thioester is transformed into phosphoryl-group-transfer potential (Figure 19.5). In the first step the coenzyme A of succinyl CoA is displaced by orthophosphate. This displacement results in another energy-rich compound, succinyl phosphate, which is a mixed anhydride. Next, a histidine residue on the enzyme acts as a moving arm, detaching the phosphoryl group from succinyl phosphate, then swinging over to ADP bound to the enzyme, and transfers the phosphoryl group to form ATP. The participation of high-energy compounds in all the steps is evidenced by the fact that the reaction is readily reversible: ΔG°′ = −3.4 kJ mol⁻¹ (−0.8 kcal mol⁻¹). The generation of ATP in a reaction in which a high-phosphoryl-transfer-potential compound (succinyl phosphate) transfers the phosphate to ADP to generate ATP is called substrate-level phosphorylation. Recall that glycolysis forms ATP with substrate-level phosphorylation reactions (Chapter 16). In Section 9, we will examine how ion gradients can be used to power ATP formation.

Succinate is subsequently oxidized to regenerate oxaloacetate.

The reactions constitute a metabolic motif that we will see again in fatty acid degradation (Chapter 27) and synthesis (Chapter 28). A methylene group (CH₂) is converted into a carbonyl group (C=O) in three steps: an oxidation, a hydration, and a second oxidation reaction. Oxaloacetate is thereby regenerated for another round of the cycle, and more energy is extracted in the form of FADH₂ and NADH.

The first step in this set of reactions, the oxidation of succinate to fumarate, is catalyzed by succinate dehydrogenase. The hydrogen acceptor is FAD rather than NAD⁺, which is used in the other three oxidation reactions in the cycle. FAD is the hydrogen acceptor in this reaction because the free-energy change is insufficient to reduce NAD⁺.

Succinate dehydrogenase differs from other enzymes in the citric acid cycle because it is embedded in the inner mitochondrial membrane in association with the electron-transport chain, which also is set in the inner mitochondrial membrane. The electron-transport chain is the link between the citric acid cycle and ATP formation (Section 9). FADH₂ produced by the oxidation of succinate does not dissociate from the enzyme, in contrast with NADH produced in other oxidation–reduction reactions. Rather, succinate dehydrogenase transfers two electrons directly from FADH₂ to coenzyme Q (CoQ). A component of the electron-transport chain, CoQ passes electrons to the ultimate acceptor, molecular oxygen, as we shall see in Section 9.

After succinate has been oxidized to fumarate, the next step is the hydration of fumarate to form l-malate, a reaction catalyzed by fumarase. Finally, malate is oxidized to form oxaloacetate. This reaction is catalyzed by malate dehydrogenase, and NAD⁺ is again the hydrogen acceptor:

The standard free energy for this reaction, unlike that for the other steps in the citric acid cycle, is significantly positive ΔG°′ = +29.7 kJ mol⁻¹, or +7.1 kcal mol⁻¹). The oxidation of malate is driven by the use of the products—oxaloacetate by citrate synthase and NADH by the electron-transport chain.

The net reaction of the citric acid cycle is

Let us review the reactions that give this stoichiometry (Figure 19.6 and Table 19.1):

Isotope-labeling studies revealed that the two carbon atoms that enter each cycle are not the ones that leave. The two carbon atoms that enter the cycle as the acetyl group are retained in the initial two decarboxylation reactions (Figure 19.6) and then remain incorporated in the four-carbon acids of the cycle. Note that succinate is a symmetric molecule. Consequently, the two carbon atoms that enter the cycle can occupy any of the carbon positions in the subsequent metabolism of the four-carbon acids. The two carbon atoms that enter the cycle as the acetyl group will be released as CO₂ in subsequent trips through the cycle. To understand why citrate is not processed as a symmetric molecule, see problems 19 and 20 at the end of the chapter.

Evidence is accumulating that the enzymes of the citric acid cycle are physically associated with one another. The close arrangement of enzymes enhances the efficiency of the citric acid cycle because a reaction product can pass directly from one active site to the next through connecting channels, a process called substrate channeling.

The key catabolic function of the citric acid cycle is the production of high-energy electrons in the form of NADH and FADH₂. As will be considered in Section 9, the electron-transport chain oxidizes the NADH and FADH₂ formed in the citric acid cycle and ultimately results in the generation of 2.5 ATP per NADH, and 1.5 ATP per FADH₂. One complete turn through the citric acid cycle generates 3 NADH molecules, 1 FADH₂ molecule, and 1 ATP molecule, for a grand total of 10 molecules of ATP. In dramatic contrast, the anaerobic glycolysis of 1 glucose molecule generates only 2 ATP molecules (and 2 lactate molecules).

Recall that molecular oxygen does not participate directly in the citric acid cycle. However, the cycle operates only under aerobic conditions because NAD⁺ and FAD can be regenerated in mitochondria only by the transfer of electrons to molecular oxygen. Glycolysis has both an aerobic and an anaerobic mode, whereas the citric acid cycle is strictly aerobic. As discussed in Chapter 16, glycolysis can proceed under anaerobic conditions because NAD⁺ is regenerated in the conversion of pyruvate into lactate or ethanol.