Energy for the Body:
Oxidative Phosphorylation

Oxidation-Reduction Reactions Experiment

 
Authors: Rachel Casiday, Carolyn Herman, and Regina Frey
Revised by: Kristin Castilo and Kit Mao

Department of Chemistry, Washington University
St. Louis, MO 63130

For information or comments on this tutorial, please contact K. Mao at mao@wustl.edu.

Key Concepts:


Satisfying The Body's Need for Energy

Every day, we build bones, move muscles, eat food, think, and perform many other activities with our bodies. All of these activities are based upon chemical reactions. However, most of these reactions are not spontaneous ( i.e., they are accompanied by a positive change in free energy, D G>0) and do not occur without some other source of free energy. Hence, the body needs some sort of free-energy currency (Figure 1). A free-energy currency molecule is one that can store and release free energy when it is needed to power a given biochemical reaction.

Figure 1

Just as purchasing transactions do not occur without monetary currency, reactions in the body do not occur without energy currency.

This tutorial will answer four questions:

1. How does the body "spend" free-energy currency to make a nonspontaneous reaction spontaneous? The body uses coupled reactions (based on thermodynamic concepts).

2. How is food used to produce the reducing agents (NADH and FADH 2 ) that can regenerate the free-energy currency? From biology, we know that the body uses glycolysis and the citric-acid cycle to generate NADH and FADH 2 .

3. How are the reducing agents (NADH and FADH 2 ) able to generate the free-energy currency molecule (ATP)? Once again, coupled reactions are key.

4. What mechanism does the body use to couple the reducing agent reactions and the generation of ATP? ATP is synthesized primarily by a two-step process consisting of an electron-transport chain and a proton gradient.  This process is based on electrochemistry and equilibrium concepts , as well as thermodynamics.

The body satisfies it's never-ending need for energy through an elegant combination of processes that illustrate the principles of thermodynamics, electrochemistry and equilibrium reactions .

How Free-Energy Currency Works

Coupled reactions are frequently used in the body to drive important biochemical processes. Separate chemical reactions may be added together to form a net reaction. The free-energy change ( D G) for the net reaction is given by the sum of the free-energy changes for the individual reactions. For example, the phosphorylation of glycerol is a necessary step in forming the phospholipids that comprise cell membranes. This step actually consists of two reactions: (1) the phosphorylation of glycerol, and (2) the dephosphorylation of ATP (the free-energy-currency molecule). The reactions may be added as shown in Equations 2-4, below:

  Glycerol + HPO42- -->   (Glycerol-3-Phosphate)2- + H2O
DGo= +9.2 kJ
(nonspontaneous)
(2)
+
ATP4- + H2O --> ADP3- + HPO42- + H+
DGo= -30.5 kJ
(spontaneous)
(3)

  Glycerol + ATP4- --> (Glycerol-3-Phosphate)2- +ADP3- + H+
DGo= -21.3 kJ
(spontaneous)
(4)

ATP is the most important "free-energy-currency" molecule in living organisms (see Figure 2, below). Adenosine triphosphate (ATP) is a useful free-energy currency because the dephosphorylation reaction is very spontaneous; i.e., it releases a large amount of free energy (30.5 kJ/mol). Thus, the dephosphorylation reaction of ATP to ADP and inorganic phosphate (Equation 3) is often coupled with nonspontaneous reactions (e.g., Equation 2) to drive them forward. The body's use of ATP as a free-energy currency is a very effective strategy to cause vital nonspontaneous reactions to occur. 

Figure 2

This is the two-dimensional (ChemDraw) structure of ATP, adenosine triphosphate. The removal of one phosphate group (green) from ATP requires the breaking of a bond (blue) and results in a large release of free energy. Removal of this phosphate group (green) results in ADP, adenosine diphosphate.

The dephosphorylation of ATP is coupled with biochemical reactions in the body ( e.g., Equations 2-4) to drive these reactions forward. In a typical cell, an ATP molecule is used within a minute of its formation.  During strenuous exercise, the rate of utilization of ATP is even higher. Hence, the supply of ATP must be regenerated. We consume food to provide energy for the body, but the majority of the energy in food is not in the form of ATP. The body utilizes energy from other nutrients in the diet to produce ATP through oxidation-reduction reactions (Figure 3).

Figure 3

This flowchart shows that the energy used by the body for its many activities ultimately comes from the chemical energy in our food. The chemical energy in our food is converted to reducing agents (NADH and FADH2). These reducing agents are then used to make ATP. ATP stores chemical energy so that it is available to the body in a readily accessible form.

How is Food Used to Make the Reducing Agents Needed for the Production of ATP?

Energy must be absorbed to make ATP . This energy is supplied by the food we eat, and then it is used to synthesize two reducing agents, NADH and FADH2 . Both of these molecules are needed to produce ATP. One of the principal energy-yielding nutrients in our diet is glucose (see structure in Table 1 in the blue box below), a simple six-carbon sugar that can be broken down by the body. When the chemical bonds in glucose are broken, free energy is released. The complete breakdown of glucose into CO2 occurs in two processes: glycolysis and the citric-acid cycle. The reactions for these two processes are shown in the blue box below.

Reactions for Glycolysis and the Citric-Acid Cycle

The first process in the breakdown of glucose is glycolysis (Equation 5), in which glucose is broken down into two three-carbon molecules known as pyruvate (showed in Table 1). The pyruvate is then converted to acetyl CoA (acetyl coenzyme A) and carbon dioxide in an intermediate step (Equation 6). In the second process, known as the citric-acid cycle (Equation 7), the three-carbon molecules are further broken down into carbon dioxide. The energy released by the breakdown of glucose (red) can be used to phosphorylate (add a phosphate group to) ADP, forming ATP (green). The net reactions for glycolysis (Equation 5) and the citric-acid cycle (Equation 7) are shown below. (Note: In the equations below, glucose and the carbon compounds into which glucose is broken are shown in red; energy-currency molecules are shown in green, and reducing agents used in the synthesis of ATP are shown in blue.)

Glycolysis
Glucose + 2 HPO42- + 2 ADP3- + 2 NAD+ —>   2  Pyruvate-  + 2 ATP4- + 2 NADH + 2 H+ + 2 H2O
(5)
     
Intermediate Step
2(Pyruvate-  + Coenzyme A + NAD+ —>    Acetyl CoA + CO2 + NADH)
(6)
     
Citric-Acid Cycle
2(Acetyl CoA + 3 NAD++ FAD + GDP3- + HPO42- + 2H2O —> 2
CO2
+ 3 NADH + FADH2   + GTP4- + 2H+ + Coenzyme A)
(7)

The structures of the important molecules in Equations 5-7 are shown in Table 1 below.

Name of Molecule

Two-Dimensional (ChemDraw) Representation
(Not to scale)

Glucose

Note: Carbon atoms are shown in red.

Pyruvate

Note: Carbon atoms (from glucose) are shown in red.

Acetyl CoA

Note: Carbon atoms from glucose are shown in red. Coenzyme A is shown in purple.

NADH

Note: The part of the molecule that participates in oxidation-reduction reactions is shown in blue.

FADH2

Note: The part of the molecule that participates in oxidation-reduction reactions is shown in blue.

Table 1

This table shows the two-dimensional representations of several important molecules in Equations 5-7.

As seen in Equations 5-7 in the blue box, glycolysis and the citric-acid cycle produce a net total of only four ATP or GTP molecules (GTP is an energy-currency molecule similar to ATP) per glucose molecule. This yield is far below the amount needed by the body for normal functioning. In fact, it is also far below the actual ATP yield for glucose in aerobic organisms (organisms that use molecular oxygen). For each glucose molecule the body processes, the body actually gains approximately 30 ATP molecules! (See Figure 4, below.)  How does the body generate ATP?

The process that accounts for the high ATP yield is known as oxidative phosphorylation . A quick examination of Equations 5-7 shows that glycolysis and the citric-acid cycle generate other products besides ATP and GTP, namely NADH and FADH2 ( blue ). These products are molecules that are oxidized ( i.e., give up electrons) spontaneously. The body uses these reducing agents (NADH and FADH2 ) in an oxidation-reduction reaction .   As you will see later in this tutorial, the free energy from these redox reactions is used to drive the production of ATP.

Figure 4

This flowchart shows the major steps involved in breaking down glucose from the diet and converting its chemical energy to the chemical energy in the phosphate bonds of ATP. This figure represents the process for aerobic (oxygen-using) organisms. Note: In this flowchart, red denotes a source of carbon atoms (originally from glucose), green denotes energy-currency molecules, and blue denotes the reducing agents that can be oxidized spontaneously.

In the discussion above, we see that glucose by itself generates only a tiny amount of ATP. However, during the breakdown of glucose, a large amount of NADH and FADH2 is produced. These reducing agents that dramatically increase the amount of ATP produced. How does this work?

How are the reducing agents (NADH and FADH2) able to generate the free-energy currency molecule (ATP)?

As discussed in an earlier section about coupling reactions, ATP is used as free-energy currency by coupling its spontaneous dephosphorylation (Equation 3) with a nonspontaneous biochemical reaction to give a net release of free energy ( i.e., a net spontaneous reaction). Coupled reactions are also used to generate ATP by phosphorylating ADP. The nonspontaneous reaction of joining ADP to inorganic phosphate to make ATP (Equation 8, below and Figure 2, above) is coupled to the oxidation reaction of NADH or FADH2 (Equation 9, below). (Recall, NADH and FADH2 are produced in glycolysis and the citric-acid cycle as described in the blue box). For simplicity, we shall henceforth discuss only the oxidation of NADH; FADH2 follows a very similar oxidation pathway.

The oxidation reaction for NADH has a larger, but negative , D G than the positive D G required for the formation of ATP from ADP and phosphate. This set of coupled reactions is so important that it has been given a special name: oxidative phosphorylation . This name emphasizes the fact that an oxidation (of NADH) reaction (Equation 9 and Figure 5, below) is being coupled to a phosphorylation (of ADP) reaction (Equation 8, below and Figure 2, above). In addition, we must consider the reduction reaction (gaining of electrons) that accompanies the oxidation of NADH. (Oxidation reactions are always accompanied by reduction reactions because an electron given up by one group must be accepted by another group.) In this case, molecular oxygen (O2) is the electron acceptor, and the oxygen is reduced to water (Equation 10, below ).

The individual reactions of interest for oxidative phosphorylation are:

phosphorylation   ADP3- + HPO42- + H+ —>   ATP4- + H2O
DGo= +30.5 kJ
(nonspontaneous)
(8)
oxidation NADH —> NAD+ + H+ +  2e-
DGo= -61.9 kJ
(spontaneous)
(9)
reduction 1/2 O2 + 2H+ + 2e-—> H2O  
DGo= -158.2 kJ
(spontaneous)
(10)

The net reaction is obtained by summing the coupled reactions, as shown in Equation 11 below.

ADP3- + HPO42- + NADH + 1/2 O2 + 2H+—> ATP4- + NAD+ + 2 H2O
DGo= -189.6 kJ
(spontaneous)
(11)

The molecular changes that occur upon oxidation of NADH are shown in Figure 5 below.

Figure 5

This is a two-dimensional (ChemDraw) representation showing the change that occurs when NADH is oxidized to NAD+. "R" represents the part of the structure that is shown in black in the drawing of NADH in Table 1. The R group does not change during the oxidation half-reaction. The molecular changes that occur upon oxidation are shown in red.

In this tutorial, we have seen that nonspontaneous reactions in the body occur by coupling them with a very spontaneous reaction (usually the ATP reaction shown in Equation 3). We have just seen that ATP is produced by coupling the phosphorylation reaction with NADH oxidation (a very spontaneous reaction). But we have not yet answered the question: by what mechanism are these reactions coupled?

Coupling Reactions in Biological Systems

Every day your body carries out many nonspontaneous reactions. As discussed earlier, if a nonspontaneous reaction is coupled to a spontaneous reaction, the coupled reactions will occur spontaneously as long as the sum of the free energies for the two reactions is negative. How is this coupling achieved in the body? Living systems couple reactions in several ways, but the most common method of coupling reactions is to carry out both reactions on the same enzyme . Consider again the phosphorylation of glycerol (Equations 2-4). Glycerol is phosphorylated by the enzyme glycerol kinase, which is found in your liver. The product of glycerol phosporylation, glycerol-3-phosphate (Equation 2), is used in the synthesis of phospholipids.

Glycerol kinase is a large protein comprised of about 500 amino acids. X-ray crystallography of the protein shows us that there is a deep groove in the protein where glycerol and ATP attach (see Figure 6, below). Because the enzyme holds the ATP and the glycerol in place, the phosphate can be transferred directly from the ATP to glycerol. Instead of two separate reactions where ATP loses a phosphate (Equation 3) and glycerol picks up a phosphate (Equation 2), the enzyme allows the phosphate to move directly from ATP to glycerol (Equation 4).

The coupling in oxidative phosphorylation uses a more complicated (and amazing!) mechanism, but the end result is the same: the reactions are linked together, the net free energy for the linked reactions is negative, and, therefore, the linked reactions are spontaneous.

Figure 6

This is a schematic representation of ATP and glycerol bound to glycerol kinase. The enzyme glycerol kinase is a dimer (consists of two identical subunits). There is a deep cleft between the subunits where ATP and glycerol bind. Since the ATP and phosphate are physically so close together when they are bound to the enzyme, the phosphate can be transferred directly from ATP to glycerol. Hence, the processes of ATP losing a phosphate (spontaneous) and glycerol gaining a phosphate (nonspontaneous) are linked together as one spontaneous process.


Questions on ATP: The Body's Free-Energy Currency (How Free-Energy Currency Works)

Acetyl phosphate
DGo = -47.3 kJ/mol
Adenosine triphosphate (ATP)
DGo = -30.5 kJ/mol
Glucose-6-phosphate
DGo = -13.8 kJ/mol
Phosphoenolpyruvate (PEP)
DGo = -61.9 kJ/mol
Phosphocreatine
DGo = -43.1 kJ/mol

Neglecting any differences in difficulty synthesizing or accessing these molecules by biological systems, rank the molecules in order of their efficiency as a free-energy currency (i.e., the amount of nonspontaneous reactions enabled per phosphate removed from a molecule of free-energy currency) from the most efficient to the least efficient.

 


Mechanism of Coupling the Oxidative-Phosphorylation Reactions

In order to couple the redox and phosphorylation reactions needed for ATP synthesis in the body, there must be some mechanism linking the reactions together. In cells, this is accomplished through an elegant proton-pumping system that occurs inside special double-membrane-bound organelles (specialized cellular components) known as mitochondria. A number of proteins are required to maintain this proton-pumping system and catalyze the oxidative and phosphorylation reactions.

Synthesis of ATP (Equation 8) is coupled with the oxidation of NADH (Equation 9) and the reduction of O2 (Equation 10). There are three key steps in this process:

  1. Electrons are transferred from NADH, through a series of electron carriers, to O2. The electron carriers are proteins embedded in the inner mitochondrial membrane. (More details about the structure of the mitochondria are presented in the next section.) (See Figure 7a.)
  2. Transfer of electrons by these carriers generates a proton (H+) gradient across the inner mitochondrial membrane. (See Figure 7b.)
  3. ATP is synthesized when H + spontaneously diffuses back across the inner mitochondrial membrane. The large positive free energy of ATP synthesis is overcome by the even larger negative free energy associated with proton flow down the concentration gradient. (See Figure 7c.)

These steps are outlined in Figure 7 below.

a. Electron Transport (Oxidation-Reduction Reactions) Through a Series of Proteins in the Inner Membrane of the Mitochondria

b. Generation of H+ (Proton) Concentration Gradient Across the Inner Mitochondrial Membrane During the Electron-Transport Process (via a Proton Pump)

c. Synthesis of ATP Using Free Energy Released From Spontaneous Diffusion of H+ Back to the Matrix Inside the Inner Mitochondrial Membrane

Figure 7

The three major steps in oxidative phosphorylation are (a) oxidation-reduction reactions involving electron transfers between specialized proteins embedded in the inner mitochondrial membrane; (b) the generation of a proton (H+) gradient across the inner mitochondrial membrane (which occurs simultaneously with step (a)); and (c) the synthesis of ATP using energy from the spontaneous diffusion of electrons down the proton gradient generated in step (b).

Note: Steps (a) and (b) show cytochrome oxidase, the final electron-carrier protein in the electron-transport chain described above. When this protein accepts an electron (green) from another protein in the electron-transport chain, an Fe(III) ion in the center of a heme group (purple) embedded in the protein is reduced to Fe(II). The coordinates for the protein were determined using x-ray crystallography, and the image was rendered using SwissPDB Viewer and POV-Ray (see References).

To generate ATP, cells use a proton-pumping system made up of proteins inside the mitochondria to generate ATP. Before we examine the details of ATP synthesis, we shall step back and look at the big picture by exploring the structure and function of the mitochondria, where oxidative phosphorylation occurs.

Structure and Function of the Mitochondria

The mitochondria (Figure 8) are where the oxidative-phosphorylation reactions occur. The mitochondria are specialized, rod-shaped oval-shaped cellular compartments (organelles) with dimensions of approximately 2 µm by 0.5 µm. (Recall that the protein ferritin has a diameter of about 80 Å, or 8 x 10-3 µm.) Mitochondria are present in virtually every cell of the body. They contain the enzymes required for the citric-acid cycle (the last steps in the breakdown of glucose), oxidative phosphorylation, and the oxidation of fatty acids.

Figure 8

This is a schematic diagram showing the membranes of the mitochondrion. The purple shapes on the inner membrane represent proteins, which are described in the section below. An enlargement of the boxed portion of the inner membrane in this figure is shown in Figure 8.

The mitochondrial membranes are crucial for this organelle's role in oxidative phosphorylation. As shown in Figure 8, mitochondria have two membranes, an inner and an outer membrane. The outer membrane is permeable to most small molecules and ions because it contains large protein channels called porins . The inner membrane is impermeable to most ions and polar molecules. The inner membrane is the site of oxidative phosphorylation . Although the membrane is mostly impermeable, it contains special H+ (proton) channels and pumps that enable the coupling of the redox reaction involving NADH and O2 (Equations 9-10) to the phosphorylation reaction of ADP (Equation 8), as described below ("Oxidation-Reduction Reactions and Proton Pumping in Oxidative Phosphorylation"). (Recall the discussion of protein channels in the "Maintaining the Body's Chemistry: Dialysis in the Kidneys" Tutorial .

As shown in Figure 8, the matrix is the space inside the inner membrane; the intermembrane space is the space between the two membranes. The matrix side of the inner membrane has a negative electrical charge relative to the intermembrane space due to an H+ gradient set up by the redox reaction (Equations 9 and 10). This charge difference is used to provide free energy (G) for the phosphorylation reaction (Equation 8).

Oxidation-Reduction Reactions and Proton Pumping in Oxidative Phosphorylation

Phosphorylation of ADP (Equation 8) is coupled to the oxidation-reduction reaction of NADH and O2 (Equations 9 and 10). Electrons are not transferred directly from NADH to O2; rather, electrons pass through a series of intermediate electron carriers in the inner membrane of the mitochondrion. Why? This allows something very important to occur: the pumping of protons across the inner membrane of the mitochondrion. As we shall see, this proton pumping that is ultimately responsible for coupling the oxidation-reduction reaction to ATP synthesis.

Two major types of mitochondrial proteins (see Figure 9, below) are required for oxidative phosphorylation to occur. Both classes of proteins are located in the inner mitochondrial membrane.

  1. The electron carriers (NADH-Q reductase, ubiquinone (Q), cytochrome reductase, cytochrome c, and cytochrome oxidase are shown in shades of purple in Figure 9 below) transport electrons in a stepwise fashion from NADH to O2.  Three of these carriers (NADH-Q reductase, cytochrome reductase, and cytochrome oxidase) are also proton pumps. These pumps simultaneously pump H+ ions (protons) from the matrix to the intermembrane space. (Proton movement from one side of the membrane to the other is shown as blue arrows in Figure 9 below.) The protons that are pumped across the membrane complete the redox reaction (Equations 9 and 10). The creation of a proton gradient across the membrane is one way of storing free energy.
  2. ATP synthetase (shown in red in Figure 9 below) allows H+ ions to diffuse back into the matrix and uses the free energy released to synthesize ATP from ADP and HPO42-. The ATP synthetase is essential for the phosphorylation to occur (Equation 8). (Proton movement from one side of the membrane to the other is shown as blue arrows in Figure 9, below.)

There are two types of electron carriers: protein complexes and mobile carriers. The three protein complexes (NADH-Q reductase (1) , cytochrome reductase (3) , and cytochrome oxidase (5) ) pump protons from the matrix to the intermembrane space. The two mobile carriers (ubiquinone (2) and cytochrome c (4) ) transfer electrons between the three proton-pumping complexes. (Gold numbers refer to the labels on each protein in Figure 9, below.) Because electrons move from one carrier to another until they are finally transferred to O2, the electron carriers (shown in Figure 9,below) are said to form an electron-transport chain.

Figure 9 is a schematic representation of the proteins involved in oxidative phosphorylation. To see an animation of oxidative phosphorylation, click on "View the Movie."

Figure 9

This is a schematic diagram illustrating the transfer of electrons from NADH, through the electron carriers in the electron transport chain, to molecular oxygen. Click on the pink button below to view a QuickTime animation of the functions of the proteins embedded in the inner mitochondrial membrane that are necessary for oxidative phosphorylation. Click the blue button below to download QuickTime 4.0 to view the movie.

NADH-Q reductase (1), cytochrome reductase (3), and cytochrome oxidase (5) are electron carriers as well as proton pumps. These use the energy gained from each electron-transfer step to move protons (H+) against a concentration gradient, from the matrix to the intermembrane space. Ubiquinone (Q) (2) and cytochrome c (Cyt C) (4) are mobile electron carriers. (Ubiquinone is not actually a protein.) All of the electron carriers are shown in purple, with lighter shades representing increasingly higher reduction potentials. Together, these electron carriers form a "chain" to transport electrons from NADH to O2. The path of the electrons is shown with the green dotted line.

ATP synthetase (red) has two components: a proton channel (allowing diffusion of protons down a concentration gradient, from the intermembrane space to the matrix), and a catalytic component to catalyze the formation of ATP.

For a more complete description of each step in oxidative phosphorylation (indicated by the gold numbers), click here.

Click here for a brief description of each of the electron carriers in the electron-transport chain. It is important to note that although NADH donates two electrons and O2 ultimately accepts four electrons, each of the carriers can only transfer one electron at a time. Hence, there are several points along the chain where electrons can be collected and dispersed. For the sake of simplicity, these points are not described in this tutorial.

In the section above, we see that the oxidation-reduction process is a series of electron transfers that occurs spontaneously and produces a proton gradient. Why are the electron transfers from one electron carrier to the next spontaneous?

What causes electrons to be transferred down the electron-transport chain?

As seen in Table 2, below, and Figure 7a, in these carriers, the species being oxidized or reduced is Fe, which is found either in an iron-sulfur (Fe-S) group or in a heme group. (Recall the heme group from the Chem 151 tutorial "Hemoglobin and the Heme Group: Metal Complexes in the Blood".) The iron in these groups is alternately oxidized and reduced between Fe(II) (reduced) or Fe(III) (oxidized) states.

Table 2 shows that the electrons are transferred through the electron-transport chain because of the difference in the reduction potential of the electron carriers. As explained in the green box below, the higher the electrical potential (e) of a reduction half reaction is, the greater the tendency is for the species to accept an electron. Hence, in the electron-transport chain, electrons are transferred spontaneously from carriers whose reduction results in a small electrical potential change to carriers whose reduction results in an increasingly larger electrical potential change.

Reduction Potentials and Relationship to Free Energy

An oxidation-reduction reaction consists of an oxidation half reaction and a reduction half reaction. Every half reaction has an electrical potential (e). By convention, all half reactions are written as reductions, and the electrical potential for an oxidation half-reaction is equal in magnitude, but opposite in sign, to the electrical potential for the corresponding reduction (i.e., the opposite reaction). The electrical potential for an oxidation-reduction reaction is calculated by

erxn = eoxidation + ereduction
(12)

For example, for the overall reaction of the oxidation of NADH paired with the reduction of O2, the potential can be calculated as shown below.

Reduction Potentials ereduction

NAD+ + 2H+ + 2e- —> NADH + H+

-0.32 V

(1/2) O2 + 2H+ + 2e- —> H2O

+0.82 V

The overall reaction is

NADH + H+ —> NAD+ + 2H+ + 2e- eoxidation = 0.32 V
(1/2) O2 + 2H+ + 2e- —> H2O ereduction = 0.82 V
net: NADH + (1/2)O2 + H+ —> H2O + NAD+ erxn = 1.14 V

The electrical potential (erxn) is related to the free energy (DG) by the following equation:

DG= -nFerxn
(13)

where n is the number of electrons transferred (in moles, from the balanced equation), and F is the Faraday constant (96,485 Coulombs/mole). (DG is given in Joules for this equation; 1 Joule = 1 Volt x 1 Coulomb.)

The overall reaction for the oxidation of NADH paired with the reduction of O2 has a negative change in free energy (DG =-220 kJ); i.e., it is spontaneous. Thus, the higher the electrical potential of a reduction half reaction, the greater the tendency for the species to accept an electron.

Just as in the box above, the electrical potential for the overall reaction (electron transfer) between two electron carriers is the sum of the potentials for the two half reactions. As long as the potential for the overall reaction is positive the reaction is spontaneous. Hence, from Table 2 below, we see that cytochrome c1 (part of the cytochrome reductase complex, #3 in Figure 9) can spontaneously transfer an electron to cytochrome c (#4 in Figure 9). The net reaction is given by Equation 16 below.

reduced cytochrome c1 —>oxidized cytochrome c1 + e-
eoxidation = -0 .220 V
(14)
oxidized cytochrome c + e-—> reduced cytochrome c
ereduction = 0.250 V
(15)
NET: reduced cyt c1 + oxidized cyt c —>oxidized cyt c1 + reduced cyt c
erxn = 0.030 V
(16)
Spontaneous

We can also see from Table 2 that cytochrome c1 cannot spontaneously transfer an electron to cytochrome b (Equation 19):

reduced cyt c1 —> oxidized cyt c1 + e-
eoxidation = - 0.220 V
(17)
oxidized cyt b + e- —> reduced cyt b
ereduction = - 0.34 V
(18)
NET: reduced cyt c1 + oxidized cyt c —>oxidized cyt c1 + reduced cyt c
erxn = - 0.56 V
(19)
NOT Spontaneous

Table 2 lists the reduction potentials for each of the cytochrome proteins (i.e., the last three steps in the electron-transport chain before the electrons are accepted by O2) involved in the electron-transport chain. Note that each electron transfer is to a cytochrome with a higher reduction potential than the previous cytochrome. As described in the box above and seen in Equations 14-19, an increase in potential leads to a decrease in DG (Equation 13), and thus the transfer of electrons through the chain is spontaneous.

Complex Name

Half Reaction

Reduction Potential

(also known as cytochrome b-c1 complex)

(3 in Figure 9)

Cytochrome b (Fe(III) center) + e- —> Cytochrome b (Fe(II) center) -0.34 V
(at pH 7, T=30oC)
Cytochrome c1 (Fe(III) center) + e- —> Cytochrome c1 (Fe(II) center) +0.220 V
(at pH 7, T=30oC)
Cytochrome c

(4 in Figure 9)

Cytochrome c (Fe(III) center) + e- —> Cytochrome c (Fe(II) center) +0.250 V
(at pH 7, T=30oC)
Cytochrome oxidase

(5 in Figure 9)

Cytochrome oxidase ( Fe(III) center) + e- —> Cytochrome oxidase   (Fe(II) center) +0.285 V
(at pH 7.4, T=25oC)

Table 2

To view the cytochrome molecules interactively using RASMOL, please click on the name of the complex to download the pdb file.

The electron-transport chain (which works because of the difference in reduction potentials) leads to a large concentration gradient for H+. As we shall see below, this huge concentration gradient leads to the production of ATP.


Questions on Electron Carriers: Steps in the Electron-Transport Chain; Reduction Potentials and Relationship to Free Energy


ATP Synthetase: Production of ATP

We have seen that the electron-transport chain generates a large proton gradient across the inner mitochondrial membrane. But recall that the ultimate goal of oxidative phosphorylation is to generate ATP to supply readily available free energy for the body. How does this occur? In addition to the electron-carrier proteins embedded in the inner mitochondrial membrane, a special protein called ATP synthetase (Figure 9, the red-colored protein) is also embedded in this membrane. ATP synthetase uses the proton gradient created by the electron-transport chain to drive the phosphorylation reaction that generates ATP (Figure 7c).

ATP synthetase is a protein that consists of two important segments: a transmembrane channel, and a catalytic component located inside the matrix. The proton-channel segment allows H+ ions to diffuse from the intermembrane space, where the concentration is high, to the matrix, where the concentration is low. Recall from the Kidney Dialysis tutorial that particles spontaneously diffuse from areas of high concentration to areas of low concentration. Since the diffusion of protons through the channel component of ATP synthetase is spontaneous, this process is accompanied by a negative change in free energy (i.e., free energy is released). The catalytic component of ATP synthetase has a site where ADP can enter. Then, using the free energy released by the spontaneous diffusion of protons through the channel segment, a bond is formed between the ADP and a free phosphate group, creating an ATP molecule. The ATP is then released from the reaction site, and a new ADP molecule can enter in order to be phosphorylated.


Questions on ATP Synthetase: Production of ATP


Summary

In this tutorial, we have learned that the ability of the body to perform daily activities is dependent on thermodynamic, equilibrium, and electrochemical concepts.   These activities, which are typically based on nonspontaneous chemical reactions, are performed by using free-energy currency. The common free-energy currency is ATP, which is a molecule that easily dephosphorylates (loses a phosphate group) and releases a large amount of free energy. In the body, the nonspontaneous reactions are coupled to this very spontaneous dephosphorylation reaction, thereby making the overall reaction spontaneous (DG < 0). As the coupled reactions occur (i.e., as the body performs daily activities), ATP is consumed and the body regenerates ATP by using energy from the food we eat (Figure 3). As seen in Figure 4, the breakdown of glucose (glycolysis) obtained from the food we eat cannot by itself generate the large amount of ATP that is needed for metabolic energy by the body. However, glycolysis and the subsequent step, the citric-acid cycle, produce two easily oxidized molecules: NADH and FADH2. These redox molecules are used in an oxidative-phosphorylation process to produce the majority of the ATP that the body uses. This oxidative-phosphorylation process consists of two steps: the oxidation of NADH (or FADH2) and the phosphorylation reaction which regenerates ATP. Oxidative phosphorylation occurs in the mitochondria, and the two reactions (oxidation of NADH or FADH2 and phosphorylation to generate ATP) are coupled by a proton gradient across the inner membrane of the mitochondria (Figure 9). As seen in Figures 7 and 9, the oxidation of NADH occurs by electron transport through a series of protein complexes located in the inner membrane of the mitochondria. This electron transport is very spontaneous and creates the proton gradient that is necessary to then drive the phosphorylation reaction that generates the ATP. Hence, oxidative-phosphorylation demonstrates that free energy can be easily transferred by proton gradients. Oxidative-phosphorylation is the primary means of generating free-energy currency for aerobic organisms, and as such it is one of the most important subjects in the study of bioenergetics (the study of energy and its chemical changes in the biological world).


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References:

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Acknowledgements:

The authors thank Dewey Holten (Washington University in St. Louis) for many helpful suggestions in the writing of this tutorial.

The development of this tutorial was supported by a grant from the Howard Hughes Medical Institute, through the Undergraduate Biological Sciences Education program, Grant HHMI# 71199-502008 to Washington University.


Copyright 1998, Washington University, All Rights Reserved.
Revised: 9/5/08