The Citric Acid Cycle

In eukaryotic cells, the pyruvate molecules produced at the end of glycolysis are transported into mitochondria, which are sites of cellular respiration. If oxygen is available, aerobic respiration will go forward. In mitochondria, pyruvate will be transformed into a two-carbon acetyl group (by removing a molecule of CO₂) that will be picked up by a carrier compound called coenzyme A (CoA), which is made from vitamin B₅. The resulting compound is called acetyl CoA. (Figure 6.3.1). Acetyl CoA can be used in a variety of ways by the cell, but its major function is to deliver the acetyl group derived from pyruvate to the citric acid (or TCA) cycle.

Like the conversion of pyruvate to acetyl CoA, the citric acid cycle in eukaryotic cells takes place in the matrix of the mitochondria. Unlike glycolysis, the citric acid cycle is a closed loop: The last part of the pathway regenerates the compound used in the first step. The eight steps of the cycle are a series of chemical reactions that converts acetyl CoA to two CO₂ molecules and in the process one ATP molecule (GTP is actually formed but contains the same amount of energy as ATP in its bonds), and reduced forms (three NADH and one FADH₂ molecules) of NAD⁺ and FAD⁺, important coenzymes in the cell. Part of this is considered an aerobic pathway (oxygen-requiring) because the NADH and FADH₂ produced must transfer their electrons to the next pathway in the system, which needs oxygen to function. If oxygen is not present, this transfer does not occur.

Two carbon atoms come into the citric acid cycle from each acetyl group. Two CO₂ molecules are released on each turn of the cycle; however, these do not contain the same carbon atoms contributed by the acetyl group on that turn of the pathway. The two acetyl-carbon atoms will eventually be released on later turns of the cycle; in this way, all six carbon atoms from the original glucose molecule will be eventually released as CO₂ in the cell. It takes two turns of the cycle to process the equivalent of one glucose molecule. Each turn of the cycle forms three high-energy NADH molecules and one high-energy FADH₂ molecule. These high-energy carriers will connect with the last portion of aerobic respiration to produce ATP molecules. One ATP (or an equivalent which is GTP) is also made in each cycle. Several of the intermediate compounds in the citric acid (TCA) cycle can be used in synthesizing non-essential amino acids; therefore, the cycle is both anabolic and catabolic in nature, and as such is called amphibolic.

Oxidative Phosphorylation

The first two pathways in glucose catabolism—glycolysis and the citric acid cycle—generates ATP. Most of the ATP generated during the aerobic catabolism of glucose, however, is not generated directly from these pathways. Rather, it derives from a process that begins with passing electrons through a series of chemical reactions to a final electron acceptor, oxygen. These reactions take place in specialized protein complexes located in the inner membrane of the mitochondria of eukaryotic organisms and on the inner part of the cell membrane of prokaryotic organisms. The energy of the electrons is harvested and used to generate an electrochemical gradient across the inner mitochondrial membrane. The potential energy of this gradient is used to generate ATP. The entirety of this process is called oxidative phosphorylation.

The electron transport chain (ETC) (Figure 6.3.2a) is the last component of aerobic respiration and is the only part of metabolism that uses oxygen. Electron transport is a series of chemical reactions where electrons are passed rapidly from one component to the next, to the endpoint of the chain where oxygen is the final electron acceptor and H₂O is produced. There are four complexes composed of proteins, labelled I through IV in Figure 6.3.2c, and the aggregation of these four complexes, together with associated mobile, accessory electron carriers, is called the electron transport chain (ETC). Many copies of the ETC are located in the inner mitochondrial membrane of eukaryotes and in the plasma membrane of prokaryotes. In each transfer of an electron through the electron transport chain, the electron loses energy, but with some transfers, the energy is stored as potential energy by using it to pump hydrogen ions (protons or H⁺) across the inner mitochondrial membrane into the intermembrane space, creating an electrochemical gradient.

Visual Connection

Cyanide inhibits cytochrome c oxidase, preventing the electrons passing to oxygen thereby preventing it being reduced to water. Following on from this the electron transport chain would no longer pump protons into the intermembrane space. The pH of the intermembrane space would increase, and ATP synthesis would stop.

Electrons from NADH and FADH₂ are passed to protein complexes in the electron transport chain. As they are passed from one complex to another (Figure 6.3.2a), the electrons lose energy, and some of which is used to pump protons from the mitochondrial matrix into the intermembrane space. In the fourth protein complex (Complex IV), the electrons are accepted by oxygen, the terminal acceptor. The oxygen with its extra electrons then combines with two protons (H⁺), further enhancing the electrochemical gradient, to form water. If there were no oxygen present in the mitochondrion, the electrons could not be removed from the system, and the entire electron transport chain would back up and stop. The mitochondria would be unable to generate new ATP in this way, and the cell will very quickly die due to a sudden lack of energy.

In the electron transport chain, the free energy from the series of reactions just described is used to pump protons (H⁺) across the membrane. The uneven distribution of these protons (H⁺) across the membrane establishes an electrochemical gradient, owing to the positive charge of these ions and a difference in concentration across the inner mitochondrial membrane.  The concentration of protons is higher in the intermembrane space than in the mitochondrial matrix.

Hydrogen ions diffuse through the inner membrane through an integral membrane protein called ATP synthase (Figure 6.3.2b). This complex protein acts as a tiny generator, turned by the force of the protons that diffuse through it, down their electrochemical gradient from the intermembrane space, where there are many mutually repelling hydrogen ions to the matrix, where there are few. The turning of the parts of this molecular machine regenerate ATP from ADP. This flow of protons across the membrane through ATP synthase is called chemiosmosis.

Chemiosmosis (Figure 6.3.2c) is used to generate 90% of the ATP made during aerobic glucose catabolism. The result of the reactions is the production of ATP from the energy of the electrons removed from hydrogen atoms. These atoms were originally part of a glucose molecule. At the end of the electron transport system, the electrons are used to reduce an oxygen molecule to oxygen ions. The extra electrons on the oxygen ions attract hydrogen ions (protons) from the surrounding medium, and water is formed. The electron transport chain and the production of ATP through chemiosmosis are collectively called oxidative phosphorylation.

ATP Yield

The number of ATP molecules generated from the catabolism of glucose varies. A source of variance is related to the shuttle of electrons across the mitochondrial membrane. The NADH generated from glycolysis cannot easily enter mitochondria. Thus, electrons are picked up on the inside of the mitochondria by either NAD⁺ or FAD⁺. Fewer ATP molecules are generated when FAD⁺ acts as a carrier. NAD⁺ is used as the electron transporter in the liver and FAD⁺ in the brain, so ATP yield depends on the tissue being considered.

Another factor that affects the yield of ATP molecules generated from glucose is that intermediate compounds in these pathways are used for other purposes. Glucose catabolism connects with the pathways that build or break down all other biochemical compounds in cells, and the result is somewhat messier than the ideal situations described thus far. For example, sugars other than glucose are fed into the glycolytic pathway for energy extraction. Other molecules that would otherwise be used to harvest energy in glycolysis or the citric acid cycle may be removed to form nucleic acids, amino acids, lipids, or other compounds. Overall, in living systems, these pathways of glucose catabolism extract about 34% of the energy contained in glucose.