B2. The Chemistry of NAD+ and FAD - Biology

NAD+ is a derivative of nicotinic acid or nicotinamide.

Figure: NAD+ is a derivative of nicotinic acid or nicotinamide.

It and its reduction product, NADH, exists in the cells as interconvertible members of a pool whose total concentration does not vary significantly with time. Hence, if carbohydrates and lipds are being oxidized by NAD+ to produce energy in the form of ATP, levels of NAD+ would begin to fall as NADH rises. A mechanism must be be present to regenerate NAD+ from NADH if oxidation is to continue. As we will see later, this happens in the muscle under anaerobic conditions (if dioxygen is lacking as when you are running a 100 or 200 m race, or if you are being chased by a saber-toothed tiger) when pyruvate + NADH react to form lactate + NAD+.

Under aerobic conditions (sufficient dioxygen available), NADH is reoxidized in the mitochondria by electron transport through a variety of mobile electron carriers, which pass electrons to dioxygen (using the enzyme complex cytochrome C oxidase) to form water.

NAD+/NADH can undergo two electron redox steps, in which a hydride is transferred from an organic molecule to the NAD+, with the electrons flowing to the positively charged nitrogen of NAD+ which serves as an electron sink. NADH does not react well with dioxgyen, since single electron transfers to/from NAD+/NADH produce free radical species which can not be stabilized effectively. All NAD+/NADH reactions in the body involve 2 electron hydride transfers.

Figure: All NAD+/NADH reactions in the body involve 2 electron hydride transfers

FAD (or flavin mononucleotide-FMN) and its reduction product, FADH2, are derivatives of riboflavin.

Figure: derivatives of riboflavin

FAD/FADH2 differ from NAD+/NADH since they are bound tightly (Kd approx 10-7 - 10-11 M) to enyzmes which use them. This is because FADH2 is susceptible to reaction with dioxygen, since FAD/FADH2 can form stable free radicals arising from single electron transfers. FAD/FADH2 can undergo 1 OR 2 electrons transfers.

Figure: FAD/FADH2 can undergo 1 OR 2 electrons transfers

FAD/FADH2 are tightly bound to enzymes so as to control the nature of the oxidizing/reducing agent that interact with them. (i.e. so dioxygen in the cell won't react with them in the cytoplasm.) If bound FAD is used to oxidize a substrate, the enzyme would be inactive in any further catalytic steps unless the bound FADH2 is reoxidized by another oxidizing agent.

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  • Illustrations integrating many different features of the reactions and their interrelationships
  • Tables listing the important system components and their function
  • Text supplementing and expanding on the illustrated facts

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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. (Recall from the experiment, "Membranes and Proteins: Dialysis, Detergents, and Proton Gradients," that the phospholipids that form cell membranes are formed from glycerol with a phosphate group and two fatty-acid chains attached.) 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:

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.

As these coupled reactions (e.g., Equations 2-4) occur, we use up ATP. 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?

To make ATP, energy must be absorbed. This energy is supplied by the food we eat, and then used to synthsize two reducing agents, NADH and FADH2 that 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. 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.)


Hence the overall reaction for the oxidation of NADH paired with the reduction of O2 has a negative change in free energy ( D G = -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 - e oxidation = - .220 V (14)
oxidized cytochrome c + e - --> reduced cytochrome c e reduction = .250 V (15)
NET: reduced cyt c1 + oxidized cyt c -->
oxidized cyt c1 + reduced cyt c
e rxn = 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 - e oxidation = - .220 V (17)
oxidized cyt b + e - --> reduced cyt b e reduction = - 0.34 V (18)
NET: reduced cyt c1 + oxidized cyt c -->
oxidized cyt c1 + reduced cyt c
e rxn = - 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 D G (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)

(4 in Figure 9)

(5 in Figure 9)

Table 2

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

Hence, 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

  • Briefly, explain why electrons travel from NADH-Q reductase, to ubiquinone (Q), to cytochrome reductase, rather than in the opposite direction.
  • One result of the transfer of electrons from NADH-Q reductase down the electron transport chain is that the concentration of protons (H + ions) in the intermembrane space is increased. Could cells move protons (H + ions) from the matrix to the intermembrane space without transporting electrons? Why or why not?

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 consisting of two important segments: a transmembrane proton 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. Thus, 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

Examples of Coenzymes

Most organisms cannot produce coenzymes naturally in large enough quantities to be effective. Instead, they are introduced to an organism in two ways:


Many coenzymes, though not all, are vitamins or derived from vitamins. If vitamin intake is too low, then an organism will not have the coenzymes needed to catalyze reactions. Water-soluble vitamins, which include all B complex vitamins and vitamin C, lead to the production of coenzymes. Two of the most important and widespread vitamin-derived coenzymes are nicotinamide adenine dinucleotide (NAD) and coenzyme A.

NAD is derived from vitamin B3 and functions as one of the most important coenzymes in a cell when turned into its two alternate forms. When NAD loses an electron, the low energy coenzyme called NAD + is formed. When NAD gains an electron, a high-energy coenzyme called NADH is formed.

NAD + primarily transfers electrons needed for redox reactions, especially those involved in parts of the citric acid cycle (TAC). TAC results in other coenzymes, such as ATP. If an organism has a NAD + deficiency, then mitochondria become less functional and provide less energy for cell functions.

When NAD + gains electrons through a redox reaction, NADH is formed. NADH, often called coenzyme 1, has numerous functions. In fact, it is considered the number one coenzyme in the human body because it is necessary for so many different things. This coenzyme primarily carries electrons for reactions and produces energy from food. For example, the electron transport chain can only begin with the delivery of electrons from NADH. A lack of NADH causes energy deficits in cells, resulting in widespread fatigue. Additionally, this coenzyme is recognized as the most powerful biological antioxidant for protecting cells against harmful or damaging substances.

Coenzyme A, also known as acetyl-CoA, naturally derives from vitamin B5. This coenzyme has several different functions. First, it is responsible for initiating fatty acid production within cells. Fatty acids form the phospholipid bilayer that comprises the cell membrane, a feature necessary for life. Coenzyme A also initiates the citric acid cycle, resulting in the production of ATP.


Non-vitamin coenzymes typically aid in chemical transfer for enzymes. They ensure physiological functions, like blood clotting and metabolism, occur in an organism. These coenzymes can be produced from nucleotides such as adenosine, uracil, guanine, or inosine.

Adenosine triphosphate (ATP) is an example of an essential non-vitamin coenzyme. In fact, it is the most widely distributed coenzyme in the human body. It transports substances and supplies energy needed for necessary chemical reactions and muscle contraction. To do this, ATP carries both a phosphate and energy to various locations within a cell. When the phosphate is removed, the energy is also released. This process is result of the electron transport chain. Without the coenzyme ATP, there would be little energy available at the cellular level and normal life functions could not occur.

Here is an example of the electron transport chain. The vitamin-derived coenzyme NADH begins the process by delivering electrons. ATP is the final resulting product:


Coenzymes are small organic molecules that link to enzymes and whose presence is essential to the activity of those enzymes. Coenzymes belong to the larger group called cofactors, which also includes metal ions cofactor is the more general term for small molecules required for the activity of their associated enzymes. The relationship between these two terms is as follows

  • Essential ions
  • Loosely bound (forming metal-activated enzymes)
  • Tightly bound (forming metalloenzymes
  • Coenzymes
  • Tightly bound prosthetic groups
  • 2 Loosely bound cosubstrates

Many coenzymes are derived from vitamins . Table 1 lists vitamins, the coenzymes derived from them, the type of reactions in which they participate, and the class of coenzyme.

Prosthetic groups are tightly bound to enzymes and participate in the catalytic cycles of enzymes. Like any catalyst , an enzyme–prosthetic group complex undergoes changes during the reaction, but before it can catalyze another reaction, it must return to its original state.

Flavin adenine dinucleotide (FAD) is a prosthetic group that participates in several intracellular oxidation -reduction reactions. During the catalytic cycle of the enzyme succinate dehydrogenase, FAD accepts two electrons from succinate, yielding fumarate as a product. Because FAD is tightly bound to the enzyme, the reaction is sometimes shown this way

where E� stands for the enzyme tightly bound to the FAD prosthetic group. In this reaction the coenzyme FAD is reduced to FADH 2 and remains tightly bound to the enzyme throughout. Before the enzyme can catalyze the oxidation of another succinate molecule, the two electrons now belonging to E�H 2 must be transferred to another electron acceptor, ubiquinone. The regenerated E� complex can then oxidize another succinate molecule.

Cosubstrates are loosely bound coenzymes that are required in stoichiometric amounts by enzymes. The molecule nicotinamide adenine dinucleotide (NAD) acts as a cosubstrate in the oxidation-reduction reaction that is catalyzed by malate dehydrogenase, one of the enzymes of the citric acid cycle.

malate + NAD + → oxaloacetate + NADH

Vitamin Coenzyme Reaction type Coenzyme class
SOURCE: Compiled from data contained in Horton, H. R., et al. (2002). Principles of Biochemistry , 3rd edition. Upper Saddle River, NJ: Prentice Hall.
B 1 (Thiamine) TPP Oxidative decarboxylation Prosthetic group
B 2 (Riboflavin) FAD Oxidation/Reduction Prosthetic group
B 3 (Pantothenate) CoA - Coenzyme A Acyl group transfer Cosubstrate
B 6 (Pyridoxine) PLP Transfer of groups to and from amino acids Prosthetic group
B 12 (Cobalamin) 5-deoxyadenosyl cobalamin Intramolecular rearrangements Prosthetic group
Niacin NAD + Oxidation/Reduction Cosubstrate
Folic acid Tetrahydrofolate One carbon group transfer Prosthetic group
Biotin Biotin Carboxylation Prosthetic group

In this reaction, malate and NAD + diffuse into the active site of malate dehydrogenase. Here NAD + accepts two electrons from malate oxaloacetate and NADH then diffuse out of the active site. The reduced NADH must then be returned to its NAD + form. For each catalytic cycle, a "new" NAD + molecule is needed if the reaction is to occur thus, stoichiometric quantities of the cosubstrate are needed. The reduced form of this coenzyme (NADH) is converted back to the oxidized form (NAD + ) via a number of simultaneously occurring processes in the cell, and the regenerated NAD + can then participate in another round of catalysis.

Coenzymes, then, are a type of cofactor. They are small organic molecules that bind tightly (prosthetic groups) or loosely (cosubstrates) to enzymes as they participate in catalysis.

What Is the Equation C6H12O6 + 6O2 --> 6CO2 + 6H2O + Energy?

The equation C6H12O6 + 6O2 --> 6CO2 + H2O + energy depicts the process of cellular respiration. This is a process in which living organisms combine food (glucose) with oxygen into energy while producing carbon dioxide and water as waste products. Since organisms can't use the energy from food directly, cellular respiration is necessary to convert the energy into a form they can use known as adenosine triphosphate (ATP).

Stage 1 of Cellular Respiration: Glycolysis

The first stage of cellular respiration is known as glycolysis or glucose splitting. Enzymes split glucose into two molecules of pyruvate. Two molecules of ATP are needed to perform glycolysis, but the process produces four molecules of ATP. That means that there is a net gain of two ATP molecules. It also produces energy-carrying molecules that are needed in later steps of the cellular respiration process.

Stage 2 of Cellular Respiration: The Krebs Cycle

The Krebs Cycle is also known as the citric acid cycle because it forms citric acid. The series of reactions that take place in the Krebs Cycle release energy and produce carbon dioxide as a waste product. Glucose is completely broken down, and all of the energy is stored in the bonds of four ATP molecules, 10 nicotinamide adenine dinucleotide (NADH( molecules, and two flavin adenine dinucleotide (FADH2) molecules.

Stage 3 of Cellular Respiration: Electron Transport

In the final stage of cellular respiration, the high-energy electrons from NADH and FADH2 move along the electron transport chains. Some of this energy is used to pump hydrogen ions across the inner membrane of the mitochondrion from the matrix into the intermembrane space. They flow back into the matrix to form ATP synthase, which produces ATP. This process can only occur in the presence of oxygen. The hydrogen ions that pass through the electron transport chain combine with oxygen to form water.

Producing ATP

The final stage of cellular respiration produces the most ATP. While two molecules of ATP are produced in each of the first two stages, the final stage produces as many as 34 more molecules of ATP. That's just from a single molecule of glucose. Not all energy produced from this process is in the form of ATP as much of the energy is released in the form of heat.

Aerobic vs. Anaerobic Respiration

Aerobic respiration occurs in the presence of oxygen, and anaerobic respiration does not rely on oxygen to take place. Most cellular respiration processes require oxygen. Glycolysis is the only stage that doesn't need oxygen to take place. When no oxygen is present, organisms break down their food in a process known as fermentation.

Role of Mitochondria

The mitochondria are known as the powerhouse of a cell because this is where cells produce energy. The mitochondria are made up of rod-shaped compartments that house the enzymes needed to break down food. Organisms can have thousands of mitochondria in each cell that are all working together to produce the energy necessary to carry out life processes.

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An oxidizing agent, or oxidant, gains electrons and is reduced in a chemical reaction. Also known as the electron acceptor, the oxidizing agent is normally in one of its higher possible oxidation states because it will gain electrons and be reduced. Examples of oxidizing agents include halogens, potassium nitrate, and nitric acid.

A reducing agent, or reductant, loses electrons and is oxidized in a chemical reaction. A reducing agent is typically in one of its lower possible oxidation states, and is known as the electron donor. A reducing agent is oxidized, because it loses electrons in the redox reaction. Examples of reducing agents include the earth metals, formic acid, and sulfite compounds.

Figure (PageIndex<1>): A reducing agent reduces other substances and loses electrons therefore, its oxidation state increases. An oxidizing agent oxidizes other substances and gains electrons therefore, its oxidation state decreases.

To help eliminate confusion, there is a mnemonic device to help determine oxidizing and reducing agents.

O xidation Is Loss and Reduction Is Gain of electrons

Example (PageIndex<1>): Identify Reducing and Oxidizing Agents

Identify the reducing and oxidizing agents in the balanced redox reaction:

  • Adenosine triphosphate (ATP) – The main energy molecule used by the cell.
  • Eukaryotes – Organisms that have eukaryotic cells, which are complex cells with a true nucleus and organelles.
  • Mitochondria – The organelle in the cells of eukaryotes that produces ATP.
  • Chloroplast – The organelle in plant cells that, in addition to mitochondria, produces ATP through photosynthesis.

1. Which organisms do not have mitochondria?
A. Bacteria
B. Animals
C. Plants
D. Fungi

2. Which component is not part of the ATP synthesis process?
A. Electron transport chain
B. Proton gradient
C. Flagella
D. Rotor and stalk of ATP synthase

3. Which part of ATP synthase is a motor?
B. F1-ATPase
C. Both
D. Neither

Coenzymes work by binding to the active side of the enzymes, the side that works in the reaction. Since enzymes and coenzymes are nonmetal organic molecules, they bind together by forming covalent bonds. The coenzymes share electrons with the enzymes, rather than lose or gain electrons. When they form this bond, they only help the reaction to occur by carrying and transferring electrons through the reaction. Coenzymes do not become integral parts of the enzymatic reaction. Instead, the covalent bonds are broken at the end of the reaction, and the coenzyme returns back to free circulation within the cell until it is used again.

Taking vitamins, whether from eating foods or in supplement form, increases the amount of coenzymes in the body. Some vitamins help the body produce coenzymes, such as folic acid and some of the B vitamins, while other vitamins directly act as coenzymes, such as vitamin C. Without vitamins, the body would be unable to produce coenzymes.

Watch the video: NAD+ and FAD+ Cofactors (January 2022).