The Citric Acid Cycle

Acetyl CoA is the starting point for the citric acid cycle (also called the Krebs cycle or the tricarboxylic acid cycle). This pathway of eight reactions completely oxidizes the two-carbon acetyl group to two molecules of carbon dioxide. The free energy released from these reactions is captured by ADP and the electron carriers NAD and FAD.

The metabolism of glucose to pyruvate is accompanied by a total drop in free energy of about 140 kcal/mol (585 kJ/mol). About a third of this energy is captured in the formation of ATP and reduced NAD (NADH + H+). Oxidizing pyruvate to acetate yields much additional free energy. Then, the citric acid cycle takes the acetyl group and essentially breaks it down to two molecules of CO₂, using the hydrogen atoms to reduce electron carriers and passing chemical free energy to those carriers in the process. The reduced carriers are oxidized in the respiratory chain which transfers an enormous amount of free energy to ATP.

The inputs to the citric acid cycle are acetate (in the form of acetyl CoA), water, and oxidized electron carriers (NAD+ and FAD).

The outputs are carbon dioxide, reduced electron carriers (NADH + H+ and FADH2), and a small amount of ATP.

Overall, for each acetyl group, the citric acid cycle removes two carbons as CO₂ and uses four pairs of hydrogen atoms to reduce electron carriers.

The citric acid cycle produces two CO₂ molecules and reduced carriers

Acetyl CoA enters the citric acid cycle from pyruvate oxidation, which has released CO₂. At the beginning of the citric acid cycle, acetyl CoA, which has two carbon atoms in its acetyl group, reacts with a four-carbon acid, oxaloacetate, to form the six-carbon compound citrate (citric acid). The remainder of the cycle consists of a series of enzyme-catalyzed reactions in which citrate is converted to a new four-carbon molecule of oxaloacetate. This new oxaloacetate can react with a second acetyl CoA, producing a second molecule of citrate and thus enabling the cycle to continue. The citric acid cycle is maintained in a steady state — that is, although the intermediate compounds in the cycle enter and leave, the concentrations of those intermediates do not change much. Also, recall that energy is released upon oxidation and stored in either ATP, FADH2 or NADH + H+. The energy temporarily stored in acetyl CoA drives the formation of citrate from oxaloacetate (reaction 1). During this reaction, the coenzyme Amolecule is removed and can be reused. In reaction 2, the citrate molecule is rearranged to form isocitrate. In reaction 3, a CO₂ molecule and two hydrogen atoms are removed, converting isocitrate to ketoglutarate. This reaction produces a large drop in free energy, some of which is stored in NADH + H+. Like the oxidation of pyruvate to acetyl CoA, reaction 4of the citric acid cycle is complex. The five-carbon -ketoglutarate molecule is oxidized to the four-carbon molecule succinate. In the process, CO₂ is given off, some of the oxidation energy is stored in NADH + H+, and some of the energy is preserved temporarily by combining succinate with CoA to form succinyl CoA. In reaction 5, some of the energy in succinyl CoA is harvested to make GTP (guanosine triphosphate) from GDP and Pi, which is another example of substrate-level phosphorylation. GTP is then used to make ATP from ADP. Free energy is released in reaction 6, in which the succinate released from succinyl CoA in reaction 5 is oxidized to fumarate. In the process, two hydrogens are transferred to an enzyme that contains the carrier FAD. After a molecular rearrangement (reaction 7), one more NAD+ reduction occurs, producing oxaloacetate from malate (reaction 8). These two reactions illustrate a common biochemical mechanism: Water (H2O) is added in reaction 7 to form an —OH group, and then the H from that —OH group is removed in reaction 8 to reduce NAD+ to NADH + H+. The final product, oxaloacetate, is ready to combine with another acetyl group from acetyl CoA and go around the cycle again. The citric acid cycle operates twice for each glucose molecule that enters glycolysis (once for each pyruvate that enters the mitochondrion). Although most of the enzymes of the citric acid cycle are located in the mitochondrial matrix, there are two exceptions: succinate dehydrogenase, which catalyzes reaction 6 and ketoglutarate dehydrogenase, which catalyzes reaction 4.

These enzymes are integral membrane proteins of the inner mitochondrial membrane. Generations of students have asked the question, “Why did this complicated system evolve to achieve the simple goal of oxidizing two acetyl groups to two molecules of CO2?” There are three reasons:

First, the cycle includes molecules that have other roles in the cell. As we will see later in this chapter, the intermediates of the citric acid cycle are themselves catabolic (breakdown) products or anabolic (synthesis) building blocks of other molecules, such as amino acids andnucleotides.

Second, the citric acid cycle is far more efficient at harvesting energy from acetyl CoA than any single reaction could be.

Third, evolution is a conservative, add-on process. It rarely operates by inventing an entirely new process.