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Energy 3: Krebs Cycle and Electron Transport Chain

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You should have a working knowledge of the following terms:

  • anaerobic respiration
  • ATP synthase
  • charge differential (voltage)
  • chemiosmosis
  • electron transport chain
  • FAD, FADH2
  • Krebs cycle
  • mitochondrial matrix
  • mitochondrion
  • oxidative phosphorylation
  • proton
  • terminal electron acceptor

Introduction and Goals

The previous tutorial concentrated on the first stages of cellular respiration (i.e., glycolysis). You should know that glycolysis produces pyruvate and some ATP. The pyruvate can be used in fermentation, but it can also be used in another manner. There are many high-energy electrons left in pyruvate. Next, you will learn how cells complete cellular respiration by oxidizing pyruvate to form carbon dioxide. You will also learn that these electrons are conveyed to an electron transport chain where they can participate in the synthesis of ATP. By the end of this tutorial you should have a basic understanding of:

  • The Krebs cycle
  • The electron transport chain
  • Chemiosmosis
  • The energy balance of cellular respiration

The Krebs Cycle

Recall, glycolysis results in a net gain of 2 ATP and 2 NADH molecules from one molecule of glucose. Keep in mind, this gain represents an effective transfer of 20 kcal of energy to ATP (about 10 kcal each) and about 80 kcal of energy to NADH (about 40 kcal each), for a total of about 100 kcal. The complete oxidation of glucose results in the release of 684 kcal of energy, therefore, there is a good amount of energy still remaining in pyruvate.

In eukaryotes, pyruvate is transported across the mitochondrial membrane and then converted to acetyl CoA (with the production of NADH and carbon dioxide). Acetyl CoA is then oxidized by a series of enzymes that make up a cyclical series of reactions known as the Krebs cycle (some textbooks refer to this as the citric acid cycle). During the Krebs cycle, electrons are removed from acetyl CoA and these electrons reduce more NAD+, along with another electron carrier, FAD. The ATP that is produced is generated via substrate-level phosphorylation, just as it was in glycolysis. Each NADH and FADH2 molecule that is formed is an important source of energy that will be used to generate ATP.


While you will not be required to memorize all of the intermediates (e.g., isocitrate, fumarate, etc.) and enzymes involved in the Krebs cycle, this figure demonstrates the overall reactions that result in the complete oxidation of acetyl CoA. You can also see where carbon dioxide is released as a by-product and where electron carriers are reduced to form NADH and FADH2. Also note that the ATP formed results from the direct transfer of a phosphate from a substrate (substrate-level phosphorylation).

Figure 1.  The Krebs Cycle.  (Click to enlarge)

This figure provides a good summary of the Krebs cycle, illustrating the main reactants (acetyl CoA, NAD+, FAD, ADP, and inorganic phosphate) and products (carbon dioxide, NADH, FADH2, and ATP) from what is actually a multistep process. 

Figure 2.  An overview of the Krebs Cycle.  (Click to enlarge)

The Role of NADH and FADH2

Glucose catabolism ends during the Krebs cycle. This does not mean that all of the energy previously contained in glucose was used in the production of ATP. In fact, only one additional ATP molecule (per molecule of pyruvate) is produced by substrate-level phosphorylation in the Krebs cycle. The balance of extracted energy is tied up in NADH and FADH2.

In the next phase of cellular respiration, the high-energy electrons within NADH and FADH2 will be passed to a set of membrane-bound enzymes in the mitochondrion, collectively referred to as the electron transport chain. These electrons will provide energy to do work. Specifically, this work will involve the movement of positively charged hydrogen atoms (H+), also known as protons.

The movement of protons across the inner mitrochondrial membrane (by the electron transport chain) creates a charge differential (voltage) that will be used to synthesize ATP. Mitochondrion structure and the origin of mitochondria will be discussed in more detail in Tutorial 24.


The Electron Transport Chain

NADH and FADH2 convey their electrons to the electron transport chain. This transport chain is composed of a number of molecules (mostly proteins) that are located in the inner membrane of the mitochondrion. Each membrane protein has a particular electronegativity (affinity for electrons). The more electronegative the molecule, the more energy required to keep the electron away from it. In this way, a slightly electronegative membrane protein will pull electrons away from reduced electron carriers. In the presence of an even-more electronegative molecule, these electrons will be oxidized from the first membrane protein, and so on. Finally the electrons reduce oxygen, and along with the addition of hydrogen ions, water is produced as a waste product. This stepwise movement, whereby an electron from one protein is transferred to another in the chain, is also reflective of the overall decrease in the amount of energy that the electron possesses. Importantly, each step has a negative delta G. Therefore with each oxidation/reduction reaction, energy is made available to do work. This work involves the movement of protons.


Oxygen is one of the most electronegative atoms. This is important because the relative change in electronegativity determines how much energy is available to do work. When oxygen acts as the terminal electron acceptor, there is a maximal amount of free energy released; hence, more protons can be transported, which means that a greater charge buildup occurs across the inner mitochondria membrane. This figure illustrates the energetic relationship between various members of the electron transport chain when oxygen serves as the electron acceptor.

 Figure 3. The Electron Transport Chain and Free Energy Change. (Click to enlarge)


During the movement of electrons through the electron transport chain, protons accumulate on the inside of the inner mitochondrial membrane. As electrons move from one member of the electron transport chain to the next, protons are transported from one side of the membrane to the other, resulting in a buildup of protons in the intermembrane space. This creates a charge differential (voltage) across the inner membrane; it is this stored energy that is actually used to synthesize ATP.

As these excess protons from the intermembrane space flow back into the mitochondrial matrix (the part of the mitochondrion enclosed within the inner membrane, which houses the enzymes and substrates for the Krebs cycle), ADP is phosphorylated to make ATP (chemiosmosis). Chemiosmosis is accomplished in the presence of the protein complex ATP synthase, which is also located in the inner mitochondrial membrane.


Note how the transfer of electrons provides the energy to move protons across the inner mitochondrial membrane. This buildup of protons in the intermembrane space creates a charge differential (voltage), and this stored energy is then used to drive the ATP synthase complex to affect the production of ATP.

Figure 4. An Overview of Electron Transport and Oxidative Phosphorylation. (Click to enlarge)

Oxidative Phosphorylation and ATP Yield

Recall, substrate-level phosphorylation was introduced in Tutorial 22. The generation of ATP from chemiosmosis is referred to as oxidative phosphorylation because oxygen's oxidative property allows a large amount of free energy to be made available for ATP synthesis.


This figure emphasizes several important concepts about cellular respiration. First, note the locations of glycolysis, the Krebs cycle, and the electron transport chain and oxidative phosphorylation. Second, note how the electron carriers transport electrons to the transport chain, and the net amount of ATP generated at each step. In particular, compare the amount of ATP generated by oxidative phosphorylation to the amount generated by substrate-level phosphorylation. The maximum net yield of 38 ATPs per molecule of glucose is merely an estimate. Much of the energy bound in a molecule of glucose is actually lost as heat during metabolism. While this heat is actually a waste product, homeotherms ("warm-blooded" animals) capitalize on this waste and use it to maintain constant body temperatures.

Figure 5. An Overview of Cellular Respiration.  (Click to enlarge)


This tutorial focused on the final steps of cellular respiration. Recall that at the end of glycolysis there is a net production of two molecules of ATP and two molecules of NADH. The ATP is produced via substrate-level phosphorylation; in this reaction, a phosphate group on an organic molecule is transferred directly (along with high-energy electrons) onto a molecule of ADP. Substrate-level phosphorylation also occurs once during the Krebs cycle.

For those organisms that completely oxidize glucose, the end product of glycolysis (pyruvate) is further oxidized by enzymes associated with the Krebs cycle (also known as the citric acid cycle and the tricarboxylic acid cycle or TCA cycle). In eukaryotes, the enzymes associated with the Krebs cycle are found in the mitochondria. Pyruvate moves into the mitochondria via specific carrier proteins located in the mitochondrial membranes (in prokaryotes, the Krebs cycle enzymes typically are not compartmentalized but are located in the same compartment as the glycolytic enzymes). Pyruvate is converted to acetyl CoA (accompanied by the evolution of CO2 and one molecule of NADH). The acetyl CoA then enters the Krebs cycle. Within the cell, the Krebs cycle organic acids are not arranged in a circle nor is there any circular arrangement of the enzymes. This pathway is termed a "cycle" (and diagrammed as a circle) because the end product becomes the first product after reacting with acetyl CoA. Acetyl CoA is not the only way that reduced carbon can enter the Krebs cycle. For example, the degradation products of some amino acids enter the Krebs cycle as organic acids (e.g., phenylalanine is converted to fumarate).
For every molecule of acetyl CoA that enters the cycle, there is a net gain of three molecules of NADH, one molecule of FADH2 (a chemical relative of NADH), and one molecule of ATP (the ATP is produced via substrate-level phosphorylation). Two molecules of CO2 are produced as a by-product. As with glycolysis, there is only a marginal gain of ATP; the majority of energy is tied up in the NADH and FADH2 molecules. How is this energy liberated? To answer this we need to consider the work that is done by NADH and FADH2.

The high-energy carriers NADH and FADH2 can themselves be oxidized by the electron transport chain. During oxidation, energy is lost by the oxidized molecule while energy is gained by the reduced molecule. The electron transport chain is composed of a series of molecules that alternatively become oxidized and reduced by one another. As these redox reactions occur, free energy is made available to do work, and that work is the movement of charged hydrogen atoms (protons) across a membrane. The electron transport chain is mostly contained within the membrane, and energetically, the electrons that pass from one molecule to the next have decreasing potential energies. The last molecule that is reduced is oxygen, which results in the generation of water. Some bacteria can use other molecules (e.g., nitrate, sulfate, or organic acids) as terminal electron acceptors, and hence can undergo cellular respiration under anaerobic conditions.

So how is ATP produced? During the movement of electrons down the electron transport chain, protons accumulate on the other side of the inner mitochondrial membrane; mitochondria have a double membrane. The accumulation of charged ions, separated by a nonconductive membrane, creates a voltage. In other words, the mitochondrion can be thought of as a battery that is charged by the electron transport chain. Also existing within the inner membrane is a complex known as the ATP synthase complex. This complex acts as a channel in which protons flow back into the mitochondrion. As these protons move back into the mitochondria, free energy is released as the charge differential decreases; the resulting energy is used to synthesize ATP. In other words, the electron transport chain charges the battery, and the ATP synthase discharges it and uses the energy to produce ATP.