Metabolism and Energy metabolism Investigation of Krebs cycle functioning

Basic principles of metabolism: catabolism, anabolism. Common pathways of proteins, carbohydrates and lipids transformation.

Studying of Krebs cycle functioning






Metabolism  is the set of life-sustaining chemical transformations within the cells of living organisms. These enzyme-catalyzed reactions allow organisms to grow and reproduce, maintain their structures, and respond to their environments. The word metabolism can also refer to all chemical reactions that occur in living organisms, including digestion and the transport of substances into and between different cells, in which case the set of reactions within the cells is called intermediary metabolism or intermediate metabolism.

The term metabolism is derived from the Greek Μεταβολισμός  "Metabolismos" for "change", or "overthrow". The history of the scientific study of metabolism spans several centuries and has moved from examining whole animals in early studies, to examining individual metabolic reactions in modern biochemistry. The first controlled experiments in human metabolism were published by Santorio Santorioin 1614 in his book Ars de statica medicina. He described how he weighed himself before and after eating, sleep, working, sex, fasting, drinking, and excreting. He found that most of the food he took in was lost through what he called "insensible perspiration".

In these early studies, the mechanisms of these metabolic processes had not been identified and a vital force was thought to animate living tissue. In the 19th century, when studying the fermentation of sugar to alcohol by yeast, Louis Pasteur concluded that fermentation was catalyzed by substances within the yeast cells he called "ferments". He wrote that "alcoholic fermentation is an act correlated with the life and organization of the yeast cells, not with the death or putrefaction of the cells."] This discovery, along with the publication by Friedrich Wöhler in 1828 of the chemical synthesis of urea, notable for being the first organic compound prepared from wholly inorganic precursors, proved that the organic compounds and chemical reactions found in cells were no different in principle than any other part of chemistry.

Metabolism is a term that is used to describe all chemical reactions involved in maintaining the living state of the cells and the organism. Metabolism can be conveniently divided into two categories:

                     Catabolism - the breakdown of molecules to obtain energy

                     Anabolism - the synthesis of all compounds needed by the cells

Anabolism is the set of constructive metabolic processes where the energy released by catabolism is used to synthesize complex molecules. In general, the complex molecules that make up cellular structures are constructed step-by-step from small and simple precursors. Anabolism involves three basic stages. Firstly, the production of precursors such as amino acids, monosaccharides,isoprenoids and nucleotides, secondly, their activation into reactive forms using energy from ATP, and thirdly, the assembly of these precursors into complex molecules such as proteins, polysaccharides, lipids and nucleic acids.

Metabolism refers to the highly integrated network of chemical reactions by which living cells grow and sustain themselves. This network is composed of two major types of pathways: anabolism and catabolism. Anabolism uses energy stored in the form of adenosine triphosphate (ATP) to build larger molecules from smaller molecules. Catabolic reactions degrade larger molecules in order to produce ATP and raw materials for anabolic reactions.

Together, these two general metabolic networks have three major functions:

(1) to extract energy from nutrients or solar energy;

(2) to synthesize the building blocks that make up the large molecules of life: proteins, fats, carbohydrates, nucleic acids, and combinations of these substances;

(3) to synthesize and degrade molecules required for special functions in the cell.

The series of products created by the sequential enzymatic steps of anabolismor catabolism are called metabolic intermediates, or metabolites. Each steprepresents a small change in the molecule, usually the removal, transfer, oraddition of a specific atom, molecule, or group of atoms that serves as a functional group, such as the amino groups (-NH2) of proteins.

Most such metabolic pathways are linear, that is, they begin with a specificsubstrate and end with a specific product. However, some pathways, such as the Krebs cycle, are cyclic. Often, metabolic pathways also have branches thatfeed into or out of them. The specific sequences of intermediates in the pathways of cell metabolism are called intermediary metabolism.

Among the many hundreds of chemical reactions there are only a few that are central to the activity of the cell, and these pathways are identical in mostforms of life.

Catabolic reactions are used to capture and save energy from nutrients, as well as to degrade larger molecules into smaller, molecular raw materials for reuse by the cell. The energy is stored in the form of energy-rich ATP, whichpowers the reactions of anabolism. The useful energy of ATP is stored in theform of a high-energy bond between the second and third phosphate groups of ATP. The cell makes ATP by adding a phosphate group to the molecule adenosinediphosphate (ADP). Therefore, ATP is the major chemical link between the energy-yielding reactions of catabolism, and the energy-requiring reactions of anabolism.

In some cases, energy is also conserved as energy-rich hydrogen atoms in thecoenzyme nicotinamide adenine dinucleotide phosphate in the reduced form of NADPH. The NADPH can then be used as a source of high-energy hydrogen atoms during certain biosynthetic reactions of anabolism.

In addition to the obvious difference in the direction of their metabolic goals, anabolism and catabolism differ in other significant ways. For example, the various degradative pathways of catabolism are convergent. That is, many hundreds of different proteins, polysaccharides, and lipids are broken down into relatively few catabolic end products. The hundreds of anabolic pathways,however, are divergent. That is, the cell uses relatively few biosynthetic precursor molecules to synthesize a vast number of different proteins, polysaccharides, and lipids.

The opposing pathways of anabolism and catabolism may also use different reaction intermediates or different enzymatic reactions in some of the steps. Forexample, there are 11 enzymatic steps in the breakdown of glucose into pyruvic acid in the liver. But the liver uses only nine of those same steps in thesynthesis of glucose, replacing the other two steps with a different set ofenzyme-catalyzed reactions. This occurs because the pathway to degradation ofglucose releases energy, while the anabolic process of glucose synthesis requires energy. The two different reactions of anabolism are required to overcome the energy barrier that would otherwise prevent the synthesis of glucose.

Another reason for having slightly different pathways is that the corresponding anabolic and catabolic routes must be independently regulated. Otherwise,if the two phases of metabolism shared the exact pathway (only in reverse) aslowdown in the anabolic pathway would slow catabolism, and vice versa.

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Some reactions can be either catabolic or anabolic, depending on the circumstances. Such reactions are called amphibolic reactions. Many of the reactions interconverting the simple molecules fall in this category.

Catabolic and anabolic pathways are interrelated in three ways:

Matter (catabolic pathways furnish the precursor compounds for anabolism. Energy (catabolic pathways furnish the energy to drive anabolism). Electrons (catabolic pathways furnish the reducing power for anabolism).

Linear pathways convert one compound through a series of intermediates to another compound. An example would be glycolysis, where glucose is converted to pyruvate.


Branched pathways may either be divergent (an intermediate can enter several linear pathways to different end products) or convergent (several precursors can give rise to a common intermediate). Biosynthesis of purines and of some amino acids are examples of divergent pathways. There is usually some regulation at the branch point. The conversion of various carbohydrates into the glycolytic pathway would be an example of convergent pathways.

In a cyclic pathway, intermediates are regenerated, and so some intermediates act in a catalytic fashion. In this illustration, the cyclic pathway carries out the net conversion of X to Z. The Tricarboxylic Acid Cycle is an example of a cyclic pathway.

A pool of compounds in equilibrium with each other provides the intermediates for converting compounds to a variety of products, depending on what is fed into the pool and what is withdrawn from the pool. The phosphogluconate pathway is an example of such a pool of intermediates. The pathway can convert glucose to CO2, hexoses to pentoses, pentoses to hexoses, pentoses to trioses, etc. depending on what the cell requires in a particular situation. NADPH as a source of reducing power for anabolic reactions is also a main product of the phosphogluconate pathway.

Organisms differ in how many of the molecules in their cells they can construct for themselves. Autotrophs such as plants can construct the complex organic molecules in cells such as polysaccharides and proteins from simple molecules like carbon dioxide and water. Heterotrophs, on the other hand, require a source of more complex substances, such as monosaccharides and amino acids, to produce these complex molecules. Organisms can be further classified by ultimate source of their energy: photoautotrophs and photoheterotrophs obtain energy from light, whereas chemoautotrophs and chemoheterotrophs obtain energy from inorganic oxidation reactions.

Metabolism is closely linked to nutrition and the availability of nutrients. Bioenergetics is a term which describes the biochemical or metabolic pathways by which the cell ultimately obtains energy. Energy formation is one of the vital components of metabolism.


Carbohydrates in metabolism

Foods supply carbohydrates in three forms: starch, sugar, and cellulose (fiber). Starches and sugars form major and essential sources of energy for humans. Fibers contribute to bulk in diet.

Body tissues depend on glucose for all activities. Carbohydrates and sugars yield glucose by digestion or metabolism.Most people consume around half of their diet as carbohydrates.


Proteins in metabolism

Proteins are the main tissue builders in the body. They are part of every cell in the body. Proteins help in cell structure, functions, haemoglobin formation to carry oxygen, enzymes to carry out vital reactions and a myriad of other functions in the body. Proteins are also vital in supplying nitrogen for DNA and RNA genetic material and energy production.


Fat in metabolism

Fats are concentrated sources of energy. They produce twice as much energy as either carbohydrates or protein on a weight basis.

Carbohydrate catabolism is the breakdown of carbohydrates into smaller units. Carbohydrates are usually taken into cells once they have been digested intomonosaccharides. Once inside, the major route of breakdown is glycolysis, where sugars such as glucose and fructose are converted into pyruvate and some ATP is generated.

 Pyruvate is an intermediate in several metabolic pathways, but the majority is converted to acetyl-CoA and fed into the citric acid cycle. Although some more ATP is generated in the citric acid cycle, the most important product is NADH, which is made from NAD+ as the acetyl-CoA is oxidized. This oxidation releases carbon dioxide as a waste product. In anaerobic conditions, glycolysis produces lactate, through the enzyme lactate dehydrogenase re-oxidizing NADH to NAD+ for re-use in glycolysis. An alternative route for glucose breakdown is the pentose phosphate pathway, which reduces the coenzyme NADPH and produces pentose sugars such asribose, the sugar component of nucleic acids.

Fats are catabolised by hydrolysis to free fatty acids and glycerol. The glycerol enters glycolysis and the fatty acids are broken down bybeta oxidation to release acetyl-CoA, which then is fed into the citric acid cycle. Fatty acids release more energy upon oxidation than carbohydrates because carbohydrates contain more oxygen in their structures.

Amino acids are either used to synthesize proteins and other biomolecules, or oxidized to urea and carbon dioxide as a source of energy. The oxidation pathway starts with the removal of the amino group by a transaminase. The amino group is fed into the urea cycle, leaving a deaminated carbon skeleton in the form of a keto acid. Several of these keto acids are intermediates in the citric acid cycle, for example the deamination of glutamate forms α-ketoglutarate. The glucogenic amino acids can also be converted into glucose, through gluconeogenesis .

Stages of catabolism

Catabolism can be broken down into 3 main stages.

Stage 1 Stage of Digestion

The large organic molecules like proteins, lipids and polysaccharides are digested into their smaller components outside cells. This stage acts on starch, cellulose or proteins that cannot be directly absorbed by the cells and need to be broken into their smaller units before they can be used in cell metabolism.

Digestive enzymes include glycoside hydrolases that digest polysaccharides into monosaccharides or simple sugars.

The primary enzyme involved in protein digestion is pepsin which catalyzes the nonspecific hydrolysis of peptide bonds at an optimal pH of 2.  In the lumen of the small intestine, the pancreas secretes zymogens of trypsin, chymotrypsin, elastase etc.  These proteolytic enzymes break the proteins down into free amino acids as well as dipeptides and tripeptides. The free amino acids as well as the di and tripeptides are absorbed by the intestinal mucosa cells which subsequently are released into the blood stream where they are absorbed by other tissues.

The amino acids and sugars are then pumped into cells by specific active transport proteins.

Stage 2 Release of energy

Once broken down these molecules are taken up by cells and converted to yet smaller molecules, usually acetyl coenzyme A (acetyl-CoA), which releases some energy.

Stage 3 - The acetyl group on the CoA is oxidised to water and carbon dioxide in the citric acid cycle and electron transport chain, releasing the energy that is stored by reducing the coenzyme nicotinamide adenine dinucleotide (NAD+) into NADH.


Carbohydrate breakdown

When complex carbohydrates are broken they form simple sugars or monosaccharides. This is taken up by the cells. Once inside these sugars undergo glycolysis, where sugars such as glucose and fructose are converted into pyruvate and some ATP is generated. Pyruvate is an intermediate in several metabolic pathways, but the majority is converted to acetyl-CoA and fed into the citric acid cycle or the Krebs cycle.

Within the citric acid cycle more ATP is generated by the monosaccharides. The most important product is NADH, which is made from NAD+ as the acetyl-CoA is oxidized. This oxidation releases carbon dioxide as a waste product.

When there is no oxygen, glycolysis produces lactate, through the enzyme lactate dehydrogenase, re-oxidizing NADH to NAD+ for re-use in glycolysis.

Glucose can also be broken down by pentose phosphate pathway, which reduces the coenzyme NADPH and produces pentose sugars such as ribose, the sugar component of nucleic acids.


Amino acid breakdown

Proteins are broken down into amino acids. Amino acids are either used to synthesize proteins and other biomolecules, or oxidized to urea and carbon dioxide as a source of energy.

In the process of oxidation, first the amino group is removed by a transaminase. The amino group is fed into the urea cycle, leaving a deaminated carbon skeleton in the form of a keto acid.

These keto acids enter the citric acid cycle. Glutamate, for example, forms α-ketoglutarate. Some of the amines may also be converted into glucose, through gluconeogenesis.


Lipid breakdown

Fats are catabolised by hydrolysis to free fatty acids and glycerol. The glycerol enters glycolysis and the fatty acids are broken down by beta oxidation to release acetyl-CoA. This acetyl co-A reaches the citric acid cycle next.

The chemical reactions of metabolism are organized into metabolic pathways. These allow the basic chemicals from nutrition to be transformed through a series of steps into another chemical, by a sequence of enzymes.

Each metabolic pathway consists of a series of biochemical reactions that are connected by their intermediates: the products of one reaction are the substrates for subsequent reactions, and so on. For example, one pathway may be responsible for the synthesis of a particular amino acid, but the breakdown of that amino acid may occur via a separate and distinct pathway. One example of an exception to this "rule" is the metabolism of glucose. Glycolysis results in the breakdown of glucose, but several reactions in the glycolysis pathway are reversible and participate in the re-synthesis of glucose (gluconeogenesis).



                     Glycolysis was the first metabolic pathway discovered:

1.                 As glucose enters a cell, it is immediately phosphorylated by ATP to glucose 6-phosphate in the irreversible first step.

2.                 In times of excess lipid or protein energy sources, certain reactions in the glycolysis pathway may run in reverse in order to produce glucose 6-phosphate which is then used for storage as glycogen or starch.

                     Metabolic pathways are often regulated by feedback inhibition.

                     Some metabolic pathways flow in a 'cycle' wherein each component of the cycle is a substrate for the subsequent reaction in the cycle, such as in the Krebs Cycle (see below).

                     Anabolic and catabolic pathways in eukaryotes often occur independently of each other, separated either physically by compartmentalization within organelles or separated biochemically by the requirement of different enzymes and co-factors.

Several distinct but linked metabolic pathways are used by cells to transfer the energy released by breakdown of fuel molecules into ATPand other small molecules used for energy (e.g. GTP, NADPH, FADH).

These pathways occur within all living organisms in some form:

1.                 Glycolysis

2.                 Aerobic respiration and/or Anaerobic respiration

3.                 Citric acid cycle / Krebs cycle (not in most obligate anaerobic organisms)

4.                 Oxidative phosphorylation (not in obligate anaerobic organisms)


Catabolism is characterized by convergence of three major routs toward a final common pathway.

Different proteins, fats and carbohydrates enter the same pathway tricarboxylic acid cycle.



Anabolism can also be divided into stages, however the anabolic pathways are characterized by divergence.

Monosaccharide synthesis begin with CO2, oxaloacetate, pyruvate or lactate. Amino acids are synthesized from acetyl CoA, pyruvate or keto acids of Krebs cycle. .

Fatty acids are constructed from acetyl CoA.

On the next stage monosaccharides, amino acids and fatty acids are used for the synthesis of polysaccharides, proteins and fats.


Compartmentation of metabolic processes permits:

- separate pools of metabolites within a cell

- simultaneous operation of opposing metabolic paths

- high local concentrations of metabolites

Example: fatty acid synthesis enzymes (cytosol), fatty acid breakdown enzymes (mitochondria).


Pyruvate Dehydrogenase


Glycolysis enzymes are located in the cytosol of cells.  Pyruvate enters the mitochondrion to be metabolized further. 

Pyruvate dehydrogenase complex is a bridge between glycolysis and aerobic metabolism citric acid cycle.

Pyruvate freely diffuses through the outer membrane of mitochon-dria through the channels formed by transmembrane proteins porins.



Mitochondrial compartments:

The mitochondrial matrix contains Pyruvate Dehydrogenase and enzymes of Krebs Cycle, plus other pathways such as fatty acid oxidation.

The mitochondrial outer membrane contains large channels, similar to bacterial porin channels, making the outer membrane leaky to ions and small molecules. 

The inner membrane is the major permeability barrier of the mitochondrion. It contains various transport catalysts, including a carrier protein that allows pyruvate to enter the matrix. It is highly convoluted, with infoldings called cristae. Embedded in the inner membrane are constituents of the respiratory chain and ATP Synthase.

Pyruvate Dehydrogenase catalyzes oxidative decarboxylation of pyruvate, to form acetyl-CoA. The overall reaction is shown below.


Pyruvate dehydrogenase complex is giant, with molecular mass ranging from 4 to 10 million daltons.

Pyruvate Dehydrogenase is a large complex containing many copies of each of three enzymes, E1, E2, and E3

The inner core of the mammalian Pyruvate Dehydrogenase complex is an icosahedral structure consisting of 60 copies of E2.





At the periphery of the complex are:

                     30 copies of E1 (itself a tetramer with subunits a2b2) and

                     12 copies of E3 (a homodimer), plus 12 copies of an E3 binding protein that links E3 to E2.


Prosthetic groups are listed below



Prosthetic Group

Pyruvate Dehydrogenase


Thiamine pyrophosphate (TPP)

Dihydrolipoyl Transacetylase



Dihydrolipoyl Dehydrogenase




Thiamine pyrophosphate (TPP) is a derivative of  thiamine (vitamin B1). Nutritional deficiency of thiamine leads to the disease beriberi. Beriberi affects especially the brain, because TPP is required for carbohydrate metabolism, and the brain depends on glucose metabolism for energy.


A proton readily dissociates from the C that is between N and S in the thiazole ring of TPP. The resulting carbanion (ylid) can attack the electron-deficient keto carbon of  pyruvate.

Lipoamide includes a dithiol that undergoes oxidation and reduction. 

The carboxyl group at the end of lipoic acid's hydrocarbon chain forms an amide bond to the side-chain amino group of a lysine residue of E2.

A long flexible arm, including hydrocarbon chains of lipoate and the lysine R-group, links the dithiol of each lipoamide to one of two lipoate-binding domains of each E2. Lipoate-binding domains are themselves part of a flexible strand of E2 that extends out from the core of the complex.

The long flexible attachment allows lipoamide functional groups to swing back and forth between E2 active sites in the core of the complex and active sites of E1 & E3 in the outer shell of the complex.

The E3 binding protein (that binds E3 to E2) also has attached lipoamide that can exchange reducing equivalents with lipoamide on E2.


FAD (Flavin Adenine Dinucleotide) is a derivative of the B-vitamin riboflavin (dimethylisoalloxazine-ribitol). The flavin ring system undergoes oxidation/reduction as shown below. Whereas NAD+ is a coenzyme that reversibly binds to enzymes, FAD is a prosthetic group, that is permanently part of the complex. 

FAD accepts and donates 2 electrons with 2 protons (2 H):

FAD + 2 e- + 2 H+ �� FADH2


Organic arsenicals are potent inhibitors of lipoamide-containing enzymes such as Pyruvate Dehydrogenase. These highly toxic compounds react with "vicinal" dithiols such as the functional group of lipoamide as shown below.


In the overall reaction, the acetic acid generated is transferred to coenzyme A.


The final electron acceptor is NAD+.

The reaction proceeds as follows:


The keto carbon of pyruvate reacts with the carbanion of TPP on E1 to yield an addition compound. The electron-pulling positively charged nitrogen of the thiazole ring promotes loss of CO2. What remains is hydroxyethyl-TPP.

The hydroxyethyl carbanion on TPP of E1 reacts with the disulfide of lipoamide on E2. What was the keto carbon of pyruvate is oxidized to a carboxylic acid, as the disulfide of lipoamide is reduced to a dithiol. The acetate formed by oxidation of the hydroxyethyl moiety is linked to one of the thiols of the reduced lipoamide as a thioester (~).

The acetate is transferred from the thiol of lipoamide to the thiol of coenzyme A, yielding acetyl CoA.

The reduced lipoamide swings over to the E3 active site. Dihydrolipoamide is reoxidized to the disulfide, as 2 e- + 2 H+ are transferred to a disulfide on E3 (disulfide interchange). 

The dithiol on E3 is reoxidized as 2 e- + 2 H+ are transferred to FAD. The resulting FADH2 is reoxidized by electron transfer to NAD+, to yield NADH + H+.

Acetyl CoA, a product of the Pyruvate Dehydrogenase reaction, is a central compound in metabolism. The "high energy" thioester linkage makes it an excellent donor of the acetate moiety.



For example, acetyl CoA functions as:

                     input to the Krebs Cycle, where the acetate moiety is further degraded to CO2.

                     donor of acetate for synthesis of fatty acids, ketone bodies, and cholesterol.


Regulation of Pyruvate Dehydrogenase complex.

Allosteric Regulation

Pyruvate dehydrogenase is a major regulatory point for entry of materials into the citric acid cycle.. The enzyme is regulated allosterically and by covalent modification.

E2 - inhibited by acetyl-CoA, activated by CoA-SH

E3 - inhibited by NADH, activated by NAD+.

ATP is an allosteric inhibitor of the complex, and AMP is an activator. The activity of this key reaction is coordinated with the energy charge, the [NAD+]/[NADH] ratio, and the ratio of acetylated to free coenzyme A.



Covalent Regulation

Part of the pyruvate dehydrogenase complex, pyruvate dehydrogenase kinase, phosphorylates three specific E1 serine residues, resulting in loss of activity of pyruvate dehydrogenase. NADH and acetyl-CoA both activate the kinase. The serines are dephosphorylated by a specific enzyme called pyruvate dehydrogenase phosphatase that hydrolyzes the phosphates from the E1 subunit of the pyruvate dehydgrogenase complex. This has the effect of activating the complex. The phosphatase is activated by Ca2+ and Mg2+. Because ATP and ADP differ in their affinities for Mg2+, the concentration of free Mg2+ reflects the ATP/ADP ratio within the mitochondrion. Thus, pyruvate dehydrogenase responds to ATP levels by being turned off when ATP is abundant and further energy production is unneeded.

In mammalian tissues at rest, much less than half of the total pyruvate dehydrogenase is in the active, nonphosphorylated form. The complex can be turned on when low ATP levels signal a need to generate more ATP. The kinase protein is an integral part of the pyruvate dehydrogenase complex, whereas the phosphatase is but loosely bound.



Within the Krebs cycle, energy in the form of ATP is usually derived from the breakdown of glucose, although fats and proteins can also be utilized as energy sources. Since glucose can pass through cell membranes, it transports energy from one part of the body to another. The Krebs cycle affects all types of life and is, as such, the metabolic pathway within the cells. This pathway chemically converts carbohydrates, fats, and proteins into carbon dioxide, and converts water into serviceable energy.

The Krebs cycle is the second stage of aerobic respiration, the first being glycolysis and last being the electron transport chain; the cycle is a series of stages that every living cell must undergo in order to produce energy. The enzymes that cause each step of the process to occur are all located in the cell's "power plant"; in animals, this power plant is the mitochondria; in plants, it is the chloroplasts; and in microorganisms, it can be found in the cell membrane. The Krebs cycle is also known as the citric acid cycle, because citric acid is the very first product generated by this sequence of chemical conversions, and it is also regenerated at the end of the cycle.

The pyruvate molecules produced during glycolysis contains  a lot of energy in the bonds between their molecules. In order to use that energy, the cell must convert it into the form of ATP. To do so, pyruvate molecules are processed through the Kreb Cycle, also known as the citric acid cycle.
(Kerbs Cycle as a drawing)


1. Prior to entering the Krebs Cycle, pyruvate must be converted into acetyl CoA. This is achieved by removing a CO2 molecule from pyruvate and then removing an electron to reduce an NAD+ into NADH. An enzyme called coenzyme A is combined with the remaini ow:

2. Citrate is formed when the acetyl group from acetyl CoA combines with oxaloacetate from the previous Krebs cycle.

3. Citrate is converted into its isomer isocitrate.


4. Isocitrate is oxidized to form the 5-carbon α-ketoglutarate. This step releases one molecule of CO2 and reduces NAD+ to NADH2+.


5. The α-ketoglutarate is oxidized to succinyl CoA, yielding CO2 and NADH2+.


The a-Ketoglutarate Dehydrogenase Complex is

Similar to pyruvate dehydrogenase complex

Same coenzymes, identical mechanisms

E1 - a-ketoglutarate dehydrogenase (with TPP)

E2 dihydrolipoyl succinyltransferase (with flexible lipoamide prosthetic group)

E3 - dihydrolipoyl dehydrogenase (with FAD)


6. Succinyl CoA releases coenzyme A and phosphorylates ADP into ATP.

In the succinyl CoA synthetase reaction, the thioester bond between HS-CoA and the succinyl group is hydrolyzed. 

 Since it is a rich in energy bond, the energy released is enough for synthesizing GTP from GDP + (P). 

 This GTP is equivalent, from the energetic point of view, to ATP. In fact, GTP can transfer the (P) group to ADP to form ATP:


 Since ATP can be produced from this reaction, without participation of the respiratory chain, this process is called Substrate Level Phosphorylation (SLP) in contrast to the Oxidative Phosphorylation (ATP synthesis using the energy released in the Electron Transport Chain).

 A few other reactions in metabolism are also coupled with ATP synthesis without participation of the respiratory chain. They are considered also SLP reactions.

7. Succinate is oxidized to fumarate, converting FAD to FADH2.

The Succinate Dehydrogenase Complex of several polypeptides, an FAD prosthetic group and iron-sulfur clusters, embedded in the inner mitochondrial membrane. Electrons are transferred from succinate to FAD and then to ubiquinone (Q) in electron transport chain. Dehydrogenation is stereospecific; only the trans isomer is formed


8. Fumarate is hydrolized to form malate.


9. Malate is oxidized to oxaloacetate, reducing NAD+ to NADH2+.

We are now back at the beginning of the Krebs Cycle. Because glycolysis produces two pyruvate molecules from one glucose, each glucose is processes through the kreb cycle twice. For each molecule of glucose, six NADH2+, two FADH2, and two ATP.



The sum of all reactions in the citric acid cycle is:

Acetyl-CoA + 3 NAD+ + FAD + GDP + Pi + 2 H2O → CoA-SH + 3 NADH + 3 H+ + FADH2 + GTP + 2 CO2

(the above reaction is equilibrated if Pi represents the H2PO4- ion, GDP the GDP2- ion and GTP the GTP3- ion).

Two carbons are oxidized to CO2, and the energy from these reactions is stored in GTP, NADH and FADH2. NADH and FADH2 are coenzymes (molecules that enable or enhance enzymes) that store energy and are utilized in oxidative phosphorylation.





Reaction type







Citrate synthase



Acetyl CoA +























Isocitrate dehydrogenase







Isocitrate dehydrogenase







α-Ketoglutarate dehydrogenase


NAD+ +

+ CO2




Succinyl-CoA synthetase

substrate level phosphorylation

GDP + Pi






Succinate dehydrogenase








Addition (H2O)





Malate dehydrogenase







                     The citric acid cycle begins with Acetyl-CoA transferring its two-carbon acetyl group to the four-carbon acceptor compound, oxaloacetate, forming citrate, a six-carbon compound.

                     The citrate then goes through a series of chemical transformations, losing first one, then a second carboxyl group as CO2.

                     Most of the energy made available by the oxidative steps of the cycle is transferred as energy-rich electrons to NAD+, forming NADH. For each acetyl group that enters the citric acid cycle, three molecules of NADH are produced.

                     Electrons are also transferred to the electron acceptor FAD, forming FADH2.

                     At the end of each cycle, the four-carbon oxaloacetate has been regenerated, and the cycle continues. Products of the first turn of the cycle are one GTP, three NADH, one FADH2, and two CO2.

                     Because two acetyl-CoA molecules are produced from each glucose molecule, two cycles are required per glucose molecule.

                     At the end of all cycles, the products are two GTP, six NADH, two FADH2, four CO2.

The detailed chemical structures have very limited medical significance, but you will find it very much easier to make sense of the other material in this course if you take the trouble to learn them! It may be helpful to follow one particular atom in acetyl CoA all the way round the cycle until it is lost as carbon dioxide, and the coloured boxes are intended to assist this process.

The oxidation of acetyl-CoA to CO2by the TCA cycle is the central process in energy metabolism. However, the TCA cycle also functions in biosynthetic pathways in which intermediates leave the cycle to be converted primarily to glucose, fatty acids, or non-essential amino acids. If TCA cycle anions are removed from the cycle they must be replaced to permit its continued function. This process is termed anaplerosis. Pyruvate carboxylase, which generates oxalacetate directly in the mitochondria, is the major anaplerotic enzyme. Conversely, 4- and 5-carbon intermediates enter the TCA cycle during the catabolism of amino acids. Because the TCA cycle cannot fully oxidize 4- and 5-carbon compounds, these intermediates must be removed from the cycle by a process termed cataplerosis.

Cataplerosis may be linked to biosynthetic processes such as gluconeogenesis in the liver and kidney cortex, fatty acid synthesis in the liver, and glyceroneogenesis in adipose tissue. Cataplerotic enzymes present in many mammalian tissues include P-enolpyruvate carboxykinase (PEPCK), glutamate dehydrogenase, aspartate aminotransferase, and citrate lyase. In this review we have evaluated the roles of anaplerosis and cataplerosis in whole body metabolism.



The TCA cycle is delicately balanced between the inflow and output of intermediates for various metabolic processes. The widely held view of the TCA cycle as a metabolic furnace needs modification in light of information supporting its role in biosynthesis. The cycle acts more as a traffic circle on a busy highway in which the flow of cars into the circle must be balanced by the flow out or the entire traffic pattern will be interrupted with disastrous consequences. In this essay we have reviewed several metabolic situations in which the two key processes, anaplerosis and cataplerosis, work together to ensure the appropriate balance of carbon flow into and out of the TCA cycle. The beauty of this fundamental biological mechanism is undeniable in its simplicity and ponderous in its complexity.






Bioenergetics is the part of biochemistry concerned with the energy involved in making and breaking of chemical bonds in the molecules found in biological organisms.

Growth, development and metabolism are some of the central phenomena in the study of biological organisms. The role of energy is fundamental to such biological processes. The ability to harness energy from a variety of metabolic pathways is a property of all living organisms. Life is dependent on energy transformations; living organisms survive because of exchange of energy within and without.

In a living organism, chemical bonds are broken and made as part of the exchange and transformation of energy. Energy is available for work (such as mechanical work) or for other processes (such as chemical synthesis and anabolic processes in growth), when weak bonds are broken and stronger bonds are made. The production of stronger bonds allows release of usable energy.

Living organisms obtain energy from organic and inorganic materials. For example, lithotrophs can oxidize minerals such as nitrates or forms of sulfur, such as elemental sulfur, sulfites, and hydrogen sulfide to produce ATP. In photosynthesis, autotrophs can produce ATP using light energy. Heterotrophs must consume organic compounds. These are mostly carbohydrates, fats, and proteins. The amount of energy actually obtained by the organism is lower than the amount present in the food; there are losses in digestion, metabolism, and thermogenesis.


Exergonic and endergonic processes. Mechanism of energy releasing and storage in the organism.


Exergonic refers to chemical reactions that proceed spontaneously from reactants to products with the release of energy. Endergonic reactions require energy input to proceed. Although the terms are often used rather loosely, they are precisely defined thermodynamic concepts based on changes in an entity called Gibbs free energy (G) accompanying reactions. Reactions in which -G decreases are exergonic, and those in which -G increases are endergonic. Exergonic reactions often involve the breakdown of organic compounds found in food, whereas endergonic reactions frequently entail synthesis of complicated molecules.

Biological metabolism contains many examples of both types, and living organisms have developed elaborate techniques for coupling the two.


Fig. 5.10

Exergonic reactions release free energy while endergonic reactions consume free energy

Although a negative -G indicates that energy must be added to the system before a reaction will occur, it tells us nothing about the rate at which it will progress. As is often the case, it may go very slowly if substantial activation energy is required to start the reaction. Living organisms have found a way around this problem by forming protein catalysts, called enzymes, that effectively reduce the amount of activation energy needed, and allow the reaction to proceed at a satisfactory rate. Enzymes do not affect the free energy of the reaction, and will not enable reactions to proceed that are not energetically feasible.

Fig. 5.12b

By coupling exergonic and endergonic reactions, organisms are able to use the available energy in food they consume to construct complex proteins, lipids, nucleic acids and carbohydrates needed for their growth and development. A well-known example involves coupling the formation of energy-rich adenosine triphosphate (ATP) from adenosine diphosphate (ADP) and phosphate (an endergonic reaction), with the transfer of hydrogen, removed from organic food materials, to oxygen (an exergonic reaction). The process is called oxidative phosphorylation. Energy stored in ATP may be used subsequently when the exergonic conversion of ATP back to ADP and phosphate is coupled with the endergonic synthesis of a needed cellular component.

Fig. 5.12c



What is macroergic bond? Examples of high energy compounds.

A bond in chemical compounds, which acts as an energy accumulator. Macroergic bond is present in some phosphorus-containing compounds in living organisms, e. g.* adenosinetriphosphate. Macroergic bonds are formed as a result of complex biochemical processes and break up when energy is released. The processes are reversible and can be repeated.

High-energy phosphate can mean one of two things:

                     The phosphate-phosphate bonds formed when compounds such as adenosine diphosphate and adenosine triphosphate are created.

                     The compounds that contain these bonds, which include the nucleoside diphosphates and nucleoside triphosphates, and the high-energy storage compounds of the muscle, the phosphagens. When people speak of a high-energy phosphate pool, they speak of the total concentration of these compounds with these high-energy bonds.


High-energy phosphate bonds are pyrophosphate bonds, acid anhydride linkages formed by taking phosphoric acid derivatives and dehydrating them. As a consequence, the hydrolysis of these bonds is exergonic under physiological conditions, releasing energy.


Energy released by high energy phosphate reactions


ΔG [kJ/mol]

ATP + H2O → ADP + Pi


ADP + H2O → AMP + Pi


ATP + H2O → AMP + PPi


PPi + H2O → 2 Pi


AMP + H2O → A + Pi



Except for PPi → 2 Pi, these reactions are, in general, not allowed to go uncontrolled in the human cell but are instead coupled to other processes needing energy to drive them to completion. Thus, high-energy phosphate reactions can:

                     provide energy to cellular processes, allowing them to run;

                     couple processes to a particular nucleoside, allowing for regulatory control of the process;

                     drive the reaction to the right, by taking a reversible process and making it irreversible.

The one exception is of value because it allows a single hydrolysis, ATP + 2H2O → AMP + PPi, to effectively supply the energy of hydrolysis of two high-energy bonds, with the hydrolysis of PPi being allowed to go to completion in a separate reaction. The AMP is regenerated to ATP in two steps, with the equilibrium reaction ATP + AMP ↔ 2ADP, followed by regeneration of ATP by the usual means, oxidative phosphorylation or other energy-producing pathways such as glycolysis.

Often, high-energy phosphate bonds are denoted by the character '~'. In this "squiggle" notation, ATP becomes A-P~P~P. The squiggle notation was invented by Fritz Albert Lipmann, who first proposed ATP as the main energy transfer molecule of the cell, in 1941. It emphasizes the special nature of these bonds.

Stryer states:

ATP is often called a high energy compound and its phosphoanhydride bonds are referred to as high-energy bonds. There is nothing special about the bonds themselves. They are high-energy bonds in the sense that free energy is released when they are hydrolyzed, for the reasons given above.

The term 'high energy' with respect to these bonds can be misleading because the negative free energy change is not due directly to the breaking of the bonds themselves. The breaking of these bonds, as with the breaking of most bonds, is an endergonic step (i.e., it absorbs energy, not releases it). The negative free energy change comes instead from the fact that the bonds formed after hydrolysis-or the phosphorylation of a residue by ATP-are lower in energy than the bonds present before hydrolysis (this includes all of the bonds involved in the reaction, not just the phosphate bonds themselves). This effect is due to a number of factors including increased resonance stabilization and solvation of the products relative to the reactants.

Besides the adenosine nucleotide phosphates, uracil, cytosine and guanine phosphates occur, too:


The triphosphate nucleosides of these compounds and those of ATP are components of RNA. They are integrated into the polymer by splitting off pyrophosphate ( = PP). The corresponding desoxyribose derivatives (dATP, dGTP, dCTP....) are necessary for DNA synthesis, where dTTP is used instead of dUTP. The terminal phosphate residues of all nucleoside di- and triphosphates are equally rich in energy. The energy set free by their hydrolysis is used for biosyntheses. They share the work equally: UTP is needed for the synthesis of polysaccharides, CTP for that of lipids and GTP for the synthesis of proteins and other molecules. These specificities are the results of the different selectivities of the enzymes, that control each of these metabolic pathways.


ATP formation

The Function of ATP

The ATP is used for many cell functions including transport work moving substances across cell membranes. It is also used for mechanical work, supplying the energy needed for muscle contraction. It supplies energy not only to heart muscle (for blood circulation) and skeletal muscle (such as for gross body movement), but also to the chromosomes and flagella to enable them to carry out their many functions. A major role of ATP is in chemical work, supplying the needed energy to synthesize the multi-thousands of types of macromolecules that the cell needs to exist.

ATP is also used as an on-off switch both to control chemical reactions and to send messages. The shape of the protein chains that produce the building blocks and other structures used in life is mostly determined by weak chemical bonds that are easily broken and remade. These chains can shorten, lengthen, and change shape in response to the input or withdrawal of energy. The changes in the chains alter the shape of the protein and can also alter its function or cause it to become either active or inactive.

 The ATP molecule can bond to one part of a protein molecule, causing another part of the same molecule to slide or move slightly which causes it to change its conformation, inactivating the molecule. Subsequent removal of ATP causes the protein to return to its original shape, and thus it is again functional. The cycle can be repeated until the molecule is recycled, effectively serving as an on and off switch. Both adding a phosphorus (phosphorylation) and removing a phosphorus from a protein (dephosphorylation) can serve as either an on or an off switch.


How is ATP Produced?

ATP is manufactured as a result of several cell processes including fermentation, respiration and photosynthesis. Most commonly the cells use ADP as a precursor molecule and then add a phosphorus to it. In eukaryotes this can occur either in the soluble portion of the cytoplasm (cytosol) or in special energy-producing structures called mitochondria. Charging ADP to form ATP in the mitochondria is called chemiosmotic phosphorylation. This process occurs in specially constructed chambers located in the mitochondrions inner membranes.


ATP-synthase converts ADP into ATP, a process called charging.

Adenosine triphosphate (ATP) is an organic molecule which stores energy used to carry out life processes. ATP is made of an adenine nucleoside, ribose sugar, and three phosphate groups. The high energy bonds between phosphate groups are broken when hydrolyzed, thus releasing energy in the system. Either one or two phosphate groups can break off, releasing Gibb's free energy, which can then be used to drive other reactions.


The molecular structure of ATP which is formed from a adenine nucleoside, ribose sugar, and three phosphate groups

ATP can be formed from bonding either adenosine monophosphate (AMP) and two inorganic phosphate groups (PPi) together or by bonding adenosine diphosphate (ADP) and one inorganic phosphate group (Pi) together. Energy is required to bond the adenosine to the phosphate groups, making it an endergonic reaction. The energy used to bond the two molecules together is then stored within covalent bonds between phosphate groups in ATP. ATP can be formed through two different endergonic processes, either through substrate-level phosphorylation or chemiosmosis. 

ATP is needed

- as a source of energy for biochemical syntheses

- for transport processes (active transport) and

- for mechanical work like movements (ciliar movements, plasma currents etc.)



How the Hydrolysis of ATP Performs Work



atp adp structure Cell Reactions and Energy


The bonds between the phosphate groups of ATPs tail can be broken by hydrolysis

Energy is released from ATP when the terminal phosphate bond is broken

This release of energy comes from the chemical change to a state of lower free energy, not from the phosphate bonds themselves

The three types of cellular work (mechanical, transport, and chemical) are powered by the hydrolysis of ATP

In the cell, the energy from the exergonic reaction of ATP hydrolysis can be used to drive an endergonic reaction

Overall, the coupled reactions are exergonic

ATP + H2O → ADP + Pi              

 Releases -30.5 kJ/mol= ΔG˚  (when one phosphate group breaks off)

ATP + H2O → AMP + PPi          

Releases -45.6 kJ/mol= ΔG˚ (when two phosphate groups break off) 



The Regeneration of ATP


ATP is a renewable resource that is regenerated by addition of a phosphate group to adenosine diphosphate (ADP)

The energy to phosphorylate ADP comes from catabolic reactions in the cell

The ATP cycle is a revolving door through which energy passes during its transfer from catabolic to anabolic pathways


Substrate-level and oxidative phosphorylation


Substrate-level phosphorylation is a type of metabolic reaction that results in the formation of adenosine triphosphate (ATP) or guanosine triphosphate (GTP) by the direct transfer and donation of a phosphoryl (PO3) group to adenosine diphosphate (ADP) or guanosine diphosphate (GDP) from a phosphorylated reactive intermediate. Note that the phosphate group does not have to come directly from the substrate. By convention, the phosphoryl group that is transferred is referred to as a phosphate group.

An alternative way to create ATP is through oxidative phosphorylation, which takes place during the process of cellular respiration, in addition to the substrate-level phosphorylation that occurs during glycolysis and the Krebs cycle. During oxidative phosphorylation, NADH is oxidized to NAD+, yielding 2.5 ATPs, and FADH2 yields 1.5 ATPs when it is oxidized. Oxidative phosphorylation uses an electrochemical or chemiosmotic gradient of protons (H+) across the inner mitochondrial membrane to generate ATP from ADP, which is a key difference from substrate-level phosphorylation.


Unlike oxidative phosphorylation, oxidation and phosphorylation are not coupled in the process of substrate-level phosphorylation, although both types of phosphorylation result in the formation of ATP and reactive intermediates are most often gained in course of oxidation processes in catabolism. However, usually most of the ATP is generated by oxidative phosphorylation in aerobic or anaerobic respiration. Substrate-level phosphorylation serves as fast source of ATP independent of external electron acceptors and respiration. This is the case for example in human erythrocytes, which have no mitochondria, and in the muscle during oxygen depression.

The main part of substrate-level phosphorylation occurs in the cytoplasm of cells as part of glycolysis and in mitochondria as part of the Krebs Cycle under both aerobic and anaerobic conditions. In the pay-off phase of glycolysis, two ATP are produced by substrate-level phosphorylation: two and only two 1,3-bisphosphoglycerate are converted to 3-phosphoglycerate by transferring a phosphate group to ADP by a kinase; two phosphoenolpyruvate are converted to pyruvate by the transfer of their phosphate groups to ADP by another kinase. The first reaction occurs after the generation of 1,3-bisphosphoglycerate from 3-phosphoglyceraldehyde and an organic phosphate via a dehydrogenase.

ATP is generated in a following separate step (key difference from oxidative phosphorylation) by transfer of the high-energy phosphate on 1,3-bisphosphoglycerate to ADP via the enzyme phosphoglycerate kinase, generating 3-phosphoglycerate. As ATP is formed of a former inorganic phosphate group, this step leads to the energy yield of glycolysis. The second substrate-level phosphorylation occurs later by means of the reaction of phosphenolpyruvate (PEP) to pyruvate via the pyruvate kinase. This reaction regenerates the ATP that has been used in the preparatory phase of glycolysis to activate glucose to glucose-6-phosphate and fructose-6-phosphate to fructose-1,6-bisphosphate, respectively.


ATP can be generated by substrate-level phosphorylation in the mitochondrial matrix, a pathway that is independent from the protonmotive force, pmf. In the mitochondrial matrix there are two reactions capable of substrate-level phosphorylation: the mitochondrial phosphoenolpyruvate carboxykinase (PEPCK), and the succinate-CoA ligase (SUCL or succinate thiokinase or succinyl-CoA synthetase). Mitochondrial PEPCK is thought to participate in the transfer of the phosphorylation potential from the matrix to cytosol and vice versa. The enzyme is a heterodimer, being composed of an invariant alpha subunit encoded by SUCLG1, and a substrate-specific beta subunit, encoded by either SUCLA2 or SUCLG2. This dimer combination results in either an ADP-forming succinate-CoA ligase (A-SUCL, EC or a GDP-forming succinate-CoA ligase (G-SUCL, EC The ADP-forming succinate-CoA ligase is potentially the only matrix enzyme generating ATP in the absence of a pmf, capable of maintaining matrix ATP levels under energy-limited conditions, such as transient hypoxia.

Another form of substrate-level phosphorylation is also seen in working skeletal muscles and the brain. Phosphocreatine is stored as a readily available high-energy phosphate supply, and the enzyme creatine phosphokinase transfers a phosphate from phosphocreatine to ADP to produce ATP. Then the ATP releases giving chemical energy.

Apart from this substrate-level phosphorylation can also be observed in fermentation, for example, heterolactic fermentation, butyric acid fermentation, and propanoic acid fermentation.


The modern views on the biological oxidation

Biological oxidation is that oxidation which occurs in biological systems to produce energy.

Oxidation can occur by:

1-Addition of oxygen (less common)

2-Removal of hydrogen (common)

3-Removal of electrons (most common)

Electrons are not stable in the free state, so their removal form a substance (oxidation) must be accompanied by their acceptance by another substance (reduction) hence the reaction is called oxidation-reduction reaction or redox reaction and the involved enzymes are called oxido-reductases


It is the affinity of a substance to accept electrons i.e. it is the potential for a substance to become reduced. Hydrogen has the lowest redoxpotential (-0.42 volt), while oxygen has the highest redoxpotential (+0.82 volt). The redoxpotentials of all other substances lie between that of hydrogen and oxygen.

Electrons are transferred from substances with low redoxpotential to substances with higher redoxpotential.This transfer of electrons is an energy yielding process and the amount of energy liberated depends on the redoxpotential difference between the electron donor and acceptor.


These enzymes catalyze oxidation-reduction reactions.

types of oxidoreductase

They are classified into five groups:

1-oxidases. 2-aerobic dehydrogenises.

3-anaerobic dehydrogenises.




1. Oxidases

An oxidase is any enzyme that catalyzes an oxidation-reduction reaction involving molecular oxygen (O2) as the electron acceptor. In these reactions, oxygen is reduced to water (H2O) or hydrogen peroxide (H2O2). The oxidases are a subclass of the oxidoreductases.




2. Aerobic Dehydrogenases(FlavoproteinLinked Oxidases).

The coenzyme of aerobic dehydrogenasesmay be:

FMN (Flavinadenine mononucleotide) as in L-amino acid oxidase.

FAD (Flavinadenine dinucleotide) as in D-amino acid oxidase, xanthineoxidase, aldehydedehydrogenaseand glucose oxidase.


3. Anaerobic Dehydrogenases

Anaerobic dehydrogenasesare further classified according to their coenzymes into:


NAD+linked anaerobic dehydrogenasese.g.

a)Cytoplasmicglycerol-3-phosphate dehydrogenase




e)β-Hydroxybutyrate dehydrogenase.


NADP linked anaerobic dehydrogenasese.g.

a)Glucose-6-phosphate dehydrogenase.



FAD linked anaerobic dehydrogenasese.g.


b)Mitochondrial glycerol-3-phosphate dehydrogenase.

c)Acy1 CoAdehydrogenase.

4. Hydroperoxidases

These enzymes use hydrogen peroxide (H2O2) as substrate changing it into water to get rid of its harmful effects.

They are further classified into peroxidasesand catalases.

Peroxidases: These enzymes need a reduced substrate as hydrogen donorperoxidase

H2O2 + XH2 (reduced substrate) ----------→ X(oxidized substrate)+ 2H2O Example:

-Glutathione peroxidasegets rid of H2O2from red cells to protect them from haemolysis

Glutathione Peroxidase

H2O2 + 2 G-S H -----------→2H2O + G-S-S-G


Catalases:These enzymes act on 2 molecules of hydrogen peroxide; one molecule is hydrogen donor & the other molecule is hydrogenaccepetor.

2H2O2 + catalase---------→ 2H2O + O2

Hydrogen peroxide is continuously produced by the action of aerobic dehydrogenasesand some oxidases. It is also produced by action of superoxide dismutase on superoxide (O2). It is removed by the action of peroxidasesand catalasesto protect cells against its harmful effects.

5. Oxygenases

These enzymes catalyze direct incorporation (addition) of oxygen into substrate.

They are further classified into dioxygenasesand monooxygenases.

A. Dioxygenases(true oxgenases)

These enzymes catalyze direct incorporation of two atoms of oxygen molecule into substrate e.g. tryptophan pyrrolase, homnogentisicacid dioxygenase, carotenaseand β-hydroxyanthranilicacid dioxygenase.


A + O2 AO2


B. Mono-oxygenases(pseudo-oxygenases; hydroxylases; mixed: function oxygenases)

AH + O2+ XH2 ------→A-OH + H2O + X

Fuctionsof cytochromeP450

Functions of microsomalcytochromeP450

1-It is important for detoxicationof xenobioticsby hydroxylation. e.g. insecticides,carcinogens,mutagensand drugs.

2-It is also important for metabolism of some drugs by hydroxylation e.g. morphine, aminopyrine, benzpyrineand aniline.

drug-H + O2+ XH2 drug-OH + H2O + X→ drug-OH + H2O + X

Function of mitochondrial cytochromeP450

1-It has a role in biosynthesis of steroid hormones from cholesterol in adrenal cortex, testis, ovary and placenta by hydroxylation

2-It has a role in biosynthesis of bile acids from cholesterol in the liver by hydroxylation at C26 by 26 hydroxylase.

3-It is important for activation of vitamin D


It is a group of hydroxylaseswhich are collectively referred to as cytochromeP450.

They are so called because their reduced forms exhibit an intense absorption band at wavelength 450 nm when complexedto carbon monoxide.

They are conjugated protein containing haeme(haemoproteins).

According to their intracellular localization they may be classified into:



It is present mainly in the microsomes of liver cells. It represents about 14% of the microsomalfraction of liver cells.

Mitochondrial cytochromeP450.

It is present in mitochondria of many tissues but it is particularly abundant in liver and steroidogenictissues as adrenal cortex, testis, ovary, placenta and kidney.


Tissue Respiration.

Tissue respiration is the release of energy, usually from glucose, in the tissues of all animals, green plants, fungi and bacteria. All these living things require energy for other processes such as growth, movement, sensitivity, and reproduction.

The most efficient form of respiration is aerobic respiration: this requires oxygen. When oxygen is not available, some organisms can respire anaerobically i.e. without air or oxygen. Yeast can respire in both ways. Yeast gets more energy from aerobic respiration, but when it runs out of oxygen it does not die. It can continue to respire anaerobically, but it does not get so much energy from the sugar. Yeast produces ethanol (alcohol) when it respires anaerobically and ultimately the ethanol will kill the yeast.

We can respire in both ways too. Normally we use oxygen, but when we are running in a race, we may not get enough oxygen into our blood, so our muscles start to respire anaerobically. Unlike yeast we produce lactic acid. Of course if we produced alcohol in our muscles it would make us drunk! Fine thing if you are running away from a predator and you end up drunk! Making lactic acid is not much better. Lactic acid causes cramp.

Glucose + Oxygen = Carbon Dioxide + Water + Energy

This word equation means: sugar and oxygen are turned into carbon dioxide and water releasing energy. You must memorise the word equation (and the balanced chemical equation if you want a grade A, B or C). Get help memorising the equations

Glucose = Carbon Dioxide + Ethanol + Energy

This word equation means: glucose is turned into carbon dioxide and ethanol releasing energy. You must memorise this word equation.


Composition of respiratory chain

NADH dehydrogenase (EC (also referred to as "NADH:quinone reductase" or "Complex I") is an enzyme located in the inner mitochondrial membrane that catalyzes the transfer of electrons from NADH to coenzyme Q (CoQ). It is the "entry enzyme" of oxidative phosphorylation in the mitochondria.



NADH Dehydrogenase is the first enzyme (Complex I) of the mitochondrial electron transport chain. There are three energy-transducing enzymes in the electron transport chain - NADH dehydrogenase (Complex I), Coenzyme Q cytochrome c reductase (Complex III), and cytochrome c oxidase (Complex IV). NADH dehydrogenase is the largest and most complicated enzyme of the electron transport chain..

The reaction of NADH dehydrogenase is:

In this process, the complex translocates four protons across the inner membrane per molecule of oxidized NADH, helping to build the electrochemical potential used to produce ATP.

The reaction can be reversed - referred to as aerobic succinate-supported NAD+ reduction - in the presence of a high membrane potential, but the exact catalytic mechanism remains unknown.

Complex I may have a role in triggering apoptosis. [5] In fact, there has been shown to be a correlation between mitochondrial activities and programmed cell death (PCD) during somatic embryo development.










All redox reactions take place in the extramembranous portion of NADH dehydrogenase. NADH initially binds to NADH dehydrogenase, and transfers two electrons to the flavin mononucleotide (FMN) prosthetic group of complex I, creating FMNH2. The electron acceptor - the isoalloxazine ring - of FMN is identical to that of FAD. The electrons are then transferred through the second prosthetic group of NADH dehydrogenase via a series of iron-sulfur (Fe-S) clusters, and finally to coenzyme Q (ubiquinone). This electron flow causes four hydrogen ions to be pumped out of the mitochondrial matrix. Ubiquinone (CoQ) accepts two electrons to be reduced to ubiquionol (CoQH2).


FADH dehydrogenase

In biochemistry, flavin adenine dinucleotide (FAD) is a redox cofactor involved in several important reactions in metabolism. FAD can exist in two different redox states, which it converts between by accepting or donating electrons. The molecule consists of a riboflavin moiety (vitamin B2) bound to the phosphate group of an ADP molecule. The flavin group is bound to ribitol, a sugar alcohol, by a carbon-nitrogen bond, not a glycosidic bond. Thus, riboflavin is not technically a nucleotide; the name flavin adenine dinucleotide is a misnomer.[1]

FAD can be reduced to FADH2, whereby it accepts two hydrogen atoms (a net gain of two electrons):

FAD FADH2 equlibrium.png

FAD reduced to FADH2

FAD (fully oxidized form, or quinone form) accepts two electrons and two protons to become FADH2 (hydroquinone form). FADH2 can then be oxidized to the semireduced form (semiquinone) FADH by donating one electron and one proton. The semiquinone is then oxidized once more by losing an electron and a proton and is returned to the initial quinone form (FAD).

FAD is an aromatic ring system, whereas FADH2 is not. This means that FADH2 is significantly higher in energy, without the stabilization that aromatic structure provides. FADH2 is an energy-carrying molecule, because, if it is oxidized, it will regain aromaticity and release all the energy represented by this stabilization.

The primary biochemical role of FADH2 in eukaryotes is to carry high-energy electrons used for oxidative phosphorylation. Its hydrogens remain in the mitochondrial matrix, whilst FAD is tightly bound to a dehydrogenase enzyme i.e. the second protein complex in the oxidative phosphorylation chain. FAD is a prosthetic group in the enzyme complex succinate dehydrogenase (complex II) that oxidizes succinate to fumarate in the eighth step of the citric acid cycle. The high-energy electrons from this oxidation are stored momentarily by reducing FAD to FADH2. FADH2 then reverts to FAD, sending its two high-energy electrons through the electron transport chain; the energy in FADH2 is enough to produce 1.5 equivalents of ATP[2] by oxidative phosphorylation. Another metabolic source of FADH2 is beta oxidation, where FAD serves as a coenzyme to acyl CoA dehydrogenase. A flavoprotein is a protein that contains a flavin moiety, this may be in the form of FAD or FMN (Flavin mononucleotide) . There are many flavoproteins besides components of the succinate dehydrogenase complex, including α-ketoglutarate dehydrogenase and a component of the pyruvate dehydrogenase complex.


These molecules are also known as coenzyme Q or mitoquinones. They are involved in electron transport in mitochondrial preparations playing an essential role in the oxidation of succinate or NADH via the cytochrome system. They serves not only as a coenzyme but also, in their reduced forms, as antioxidants. They are synthesized de novo in all animal tissues and cannot thus be regarded as vitamins. Ubiquinones are present in all aerobic organisms, plants, animals (the name ubiquinone was proposed with reference to their ubiquitous occurrence) and bacteria, but are absent from Gram-positive eubacteria and the archaebacteria. They were discovered by the Morton's group in animal fat but their quinonoid structure was revealed by Crane two years later in extracts from beef heart mitochondria.

The compound had a 2,3-dimethoxy-5-methylbenzoquinone nucleus and a side chain of 10 isoprenoid units and was referred to as coenzyme Q 10 . Later, homologues with 6, 7, 8 and 9 units were isolated from other organisms, bacteria or higher organisms. The main form in man has 10 units but in rat has 9 units. Another system of nomenclature is used: ubiquinone(x) in which x designates the total number of carbon atoms in the side chain, it can be a multiple of 5.


Ubiquinones accept one electron and are transformed into semiquinone radicals (UQH) or two electrons to give ubiquinol (UQH2)


Coenzyme Q is reducible by sodium dithionite or borohydride to its hydroquinone form, and can in turn be reoxidized to the quinone by Ag2O or more slowly by oxygen. The absorption spectra of the two forms are shown below. The quinone form has a strong absorption band at 275 nm which disappears in the reduced form.

Cytochromes are, in general, membrane-bound (i.e. inner mitochondrial membrane) hemeproteins containing heme groups and are primarily responsible for the generation of ATP via electron transport.

They are found either as monomeric proteins (e.g., cytochrome c) or as subunits of bigger enzymatic complexes that catalyze redox reactions.


Cytochromes were initially described in 1884 by MacMunn as respiratory pigments (myohematin or histohematin).[1] In the 1920s, Keilin rediscovered these respiratory pigments and named them the cytochromes, or cellular pigments, and classified these heme proteins, on the basis of the position of their lowest energy absorption band in the reduced state, as cytochromes a (605 nm), b (~565 nm), and c (550 nm). The UV-visible spectroscopic signatures of hemes are still used to identify heme type from the reduced bis-pyridine-ligated state, i.e., the pyridine hemochrome method. Within each class, cytochrome a, b, or c, early cytochromes are numbered consecutively, e.g. cyt c, cyt c1, and cyt c2, with more recent examples designated by their reduced state R-band maximum, e.g. cyt c559.[2]

Structure and function

The heme group is a highly-conjugated ring system (which allows its electrons to be very mobile) surrounding a metal ion, which readily interconverts between the oxidation states. For many cytochromes, the metal ion present is that of iron, which interconverts between Fe2+ (reduced) and Fe3+ (oxidised) states (electron-transfer processes) or between Fe2+ (reduced) and Fe3+ (formal, oxidized) states (oxidative processes). Cytochromes are, thus, capable of performing oxidation and reduction. Because the cytochromes (as well as other complexes) are held within membranes in an organized way, the redox reactions are carried out in the proper sequence for maximum efficiency.

In the process of oxidative phosphorylation, which is the principal energy-generating process undertaken by organisms, other membrane-bound and -soluble complexes and cofactors are involved in the chain of redox reactions, with the additional net effect that protons (H+) are transported across the mitochondrial inner membrane. The resulting transmembrane proton gradient ([protonmotive force]) is used to generate ATP, which is the universal chemical energy currency of life. ATP is consumed to drive cellular processes that require energy (such as synthesis of macromolecules, active transport of molecules across the membrane, and assembly of flagella).



Several kinds of cytochrome exist and can be distinguished by spectroscopy, exact structure of the heme group, inhibitor sensitivity, and reduction potential.

Three types of cytochrome are distinguished by their prosthetic groups:



Prosthetic group

Cytochrome a

heme a

Cytochrome b

heme b

Cytochrome d

tetrapyrrolic chelate of iron

The definition of cytochrome c is not defined in terms of the heme group. There is no "cytochrome e," but there is a cytochrome f, which is often considered a type of cytochrome c.

In mitochondria and chloroplasts, these cytochromes are often combined in electron transport and related metabolic pathways:




a and a3

Cytochrome c oxidase ("Complex IV") with electrons delivered to complex by soluble cytochrome c (hence the name)

b and c1

Coenzyme Q - cytochrome c reductase ("Complex III")

b6 and f

Plastoquinolplastocyanin reductase


A completely distinct family of cytochromes is known as the cytochrome P450 oxidases, so named for the characteristic Soret peak formed by absorbance of light at wavelengths near 450 nm when the heme iron is reduced (with sodium dithionite) and complexed to carbon monoxide. These enzymes are primarily involved in steroidogenesis and detoxification.


Respiratory chain


The system of mitochondrial enzymes and redox carrier molecules which ferry reducing equivalents from substrates to oxygen are collectively known as the electron transport system, or the respiratory chain. This system captures the free energy available from substrate oxidation so that it may later be applied to the synthesis of ATP. Many respiratory chain components were first identified in crude homogenates through their spectral properties, which frequently change when a carrier is oxidised or reduced. Fractionation of mitochondria in the presence of mild detergents or chaotropic salts dissected the respiratory chain into four large multi-subunit complexes containing the principal respiratory carriers, named Complex 1 to Complex 4.

These substantial protein "icebergs" float in the sheet of inner membrane lipids, often presenting one face to the mitochondrial matrix and another to the inter - membrane space. Many of their components have now been isolated in a relatively pure form. Other membrane bound enzymes such as the energy linked transhydrogenase (ELTH) are also present which fulfil ancillary roles.


The main components participate in the approximate order of their redox potentials, and the bulky complexes are linked by low molecular weight mobile carriers which ferry the reducing equivalents from one complex to the next. Except for succinate dehydrogenase (complex 2) all these complexes pump protons from the matrix space into the cytosol as they transfer reducing equivalents (either hydrogen atoms or electrons) from one carrier to the next. The diagram above shows the flow of reducing equivalents in purple, and movement of the positively charged protons in red. Proton pumping is an arduous task which creates substantial pH and electrical gradients across the mitochondrial inner membrane. These protons eventually re-enter the matrix space via the F1 ATPase, driving the synthesis of ATP as they return.

The number of protons and the number of positive charges crossing the inner membrane need not necessarily agree for each individual transmembrane protein, although the accounts must balance for the whole ensemble. This discrepancy is illustrated on the diagram above, and is explained in greater detail below.

Electrons flow through the electron transport chain to molecular oxygen; during this flow, protons are moved across the inner membrane from the matrix to the intermembrane space. This model for ATP synthesis is called the chemiosmotic mechanism, or Mitchell hypothesis. Peter Mitchell, a British biochemist, essentially by himself and in the face of contrary opinion, proposed that the mechanism for ATP synthesis involved the coupling between chemical energy (ATP) and osmotic potential (a higher concentration of protons in the intermembrane space than in the matrix). The inner membrane of the mitochondrion is tightly packed with cytochromes and proteins capable of undergoing redox changes. There are four major protein-membrane complexes.


Complex I and Complex II

Complex I and Complex II direct electrons to coenzyme Q. Complex I, also called NADH-coenzyme Q reductase, accepts electrons from NADH. The NADH releases a proton and two electrons. The electrons flow through a flavoprotein containing FMN and an iron-sulfur protein. First, the flavin coenzyme (flavin mononucleotide) and then the iron-sulfur center undergo cycles of reduction and then oxidation, transferring their electrons to a quinone molecule, coenzyme Q (see Figure ).

Complex I is capable of transferring protons from the matrix to the intermembrane space while undergoing these redox cycles. One possible source of the protons is the release of a proton from NADH as it is oxidized to NAD, although this is not the only explanation. Apparently, conformational changes in the proteins of Complex I also are involved in the mechanism of proton translocation during electron transport.


Complex II, also known as succinate-coenzyme Q reductase, accepts electrons from succinate formed during the TCA cycle. Electrons flow from succinate to FAD (the flavin-adenine dinucleotide) coenzyme, through an iron-sulfur protein and a cytochrome b550 protein (the number refers to the wavelength where the protein absorbs), and to coenzyme Q. No protons are translocated by Complex II. Because translocated protons are the source of the energy for ATP synthesis, this means that the oxidation of a molecule of FADH2 inherently leads to less ATP synthesized than does the oxidation of a molecule of NADH. This experimental observation also fits with the difference in the standard reduction potentials of the two molecules. The reduction potential of FAD is -0.22 V, as opposed to -0.32 V for NAD.

Coenzyme Q is capable of accepting either one or two electrons to form either a semiquinone or hydroquinone form. Coenzyme Q is not bound to a protein; instead it is a mobile electron carrier and can float within the inner membrane, where it can transfer electrons from Complex I and Complex II to Complex III.

Complex III is also known as coenzyme Q-cytochrome c reductase. It accepts electrons from reduced coenzyme Q, moves them within the complex through two cytochromes b, an iron-sulfur protein, and cytochrome c1. Electron flow through Complex II transfers proton(s) through the membrane into the intermembrane space. Again, this supplies energy for ATP synthesis. Complex III transfers its electrons to the heme group of a small, mobile electron transport protein, cytochrome c.

Cytochrome c transfers its electrons to the final electron transport component, Complex IV, or cytochrome oxidase. Cytochrome oxidase transfers electrons through a copper-containing protein, cytochrome a, and cytochrome a3, and finally to molecular oxygen. The overall pathway for electron transport is therefore:


The number n is a fudge factor to account for the fact that the exact stoichiometry of proton transfer isn't really known. The important point is that more proton transfer occurs from NADH oxidation than from FADH2 oxidation.


Chemiosmotic hypothesis

A theory postulated by the biochemist Peter Mitchell in 1961 to describe ATP synthesis by way of a proton electrochemical coupling is called chemiosmotic hypothesis.

Accordingly, hydrogen ions (protons) are pumped from the mitochondrial matrix to the intermembrane space via the hydrogen carrier proteins while the electrons are transferred along the electron transport chain in the mitochondrial inner membrane. As the hydrogen ions accumulate in the intermembrane space, an energy-rich proton gradient is established. As the proton gradient becomes sufficiently intense the hydrogen ions tend to diffuse back to the matrix (where hydrogen ions are less) via the ATP synthase (a transport protein). As the hydrogen ions diffuse (through the ATP synthase) energy is released which is then used to drive the conversion of ADP to ATP (by phosphorylation).



Chemiosmotic Hypothesis in a simple form

In the 1960s, ATP was known to be the energy currency of life, but the mechanism by which ATP was created in the mitochondria was assumed to be by substrate-level phosphorylation. Mitchell's chemiosmotic hypothesis was the basis for understanding the actual process of oxidative phosphorylation. At the time, the biochemical mechanism of ATP synthesis by oxidative phosphorylation was unknown.

Mitchell realised that the movement of ions across an electrochemical membrane potential could provide the energy needed to produce ATP. His hypothesis was derived from information that was well known in the 1960s. He knew that living cells had a membrane potential; interior negative to the environment. The movement of charged ions across a membrane is thus affected by the electrical forces (the attraction of positive to negative charges). Their movement is also affected by thermodynamic forces, the tendency of substances to diffuse from regions of higher concentration. He went on to show that ATP synthesis was coupled to this electrochemical gradient.

His hypothesis was confirmed by the discovery of ATP synthase, a membrane-bound protein that uses the potential energy of the electrochemical gradient to make ATP.

The passage back occurs via a specific proton channel. This passage is coupled to ATP-synthesis, using the potential energy of the proton gradient for the formation of the third phosphate bond of ATP.

We can now calculate the end result of glucose degradation: the oxidation is coupled to a decrease of the free energy; 686 kcal/mol (= 2881 kJ/mol) are obtained by the complete oxidation of glucose. How much of this energy can the cell use?

1.                 Six mol ATP per mol glucose are generated (substrate chain phosphorylation). This is because all steps after the breaking down of fructose-1,6-phosphate have to be counted twice (once for each of the two resulting C3 molecules), so it is 3 x 2 ATPs. Of these six ATPs, two are needed to start glycolysis. That leaves four.

2.                 During the course of glycolysis up to acetyl-CoA, 2 x 2 NADH + H+ are generated. An additional 3 x 2 NADH + H+ and 1 x 2 FADH2 are produced in the citric acid cycle. One NADH + H+ gives three, one FADH2 two ATPs when fed into the respiratory chain. This sums up to 34 ATPs plus the 4 ATPs of glycolysis. A total of 38 mol ATP are thus gained by the cell's degradation of one mol glucose. Since each energy-rich bond of ATP contains 7.3 kcal/mol (= -30.6 kJ/mol), the 38 ATP equal 277 kcal/mol (ca 1163 kJ/mol). This is 40.6% of the theoretically possible gain. The other 59.4 percent are set free as heat. This is a very high percentage compared to the gain of technical machines like steam or petrol engines that is around or below 20 percent.

The ATP synthase enzymes have been remarkably conserved through evolution. The bacterial enzymes are essentially the same in structure and function as those from mitochondria of animals, plants and fungi, and the chloroplasts of plants. The early ancestory of the enzyme is seen in the fact that the Archaea have an enzyme which is clearly closely related, but has significant differences from the Eubacterial branch. The H+-ATP-ase found in vacuoles of the eukaryote cell cytoplasm is similar to the archaeal enzyme, and is thought to reflect the origin from an archaeal ancestor.

In most systems, the ATP synthase sits in the membrane (the "coupling" membrane), and catalyses the synthesis of ATP from ADP and phosphate driven by a flux of protons across the membrane down the proton gradient generated by electron transfer. The flux goes from the protochemically positive (P) side (high proton electrochemical potential) to the protochemically negative (N) side. The reaction catalyzed by ATP synthase is fully reversible, so ATP hydrolysis generates a proton gradient by a reversal of this flux. In some bacteria, the main function is to operate in the ATP hydrolysis direction, using ATP generated by fermentative metabolism to provide a proton gradient to drive substrate accumulation, and maintain ionic balance.


ADP + Pi + nH+P <=> ATP + nH+N

                     the free energy change (DG) associated with synthesis of ATP under cellular conditions (the free energy required)

Because the structures seen in EM, the subunit composition, and the sequences of the subunits appeared to be so similar, it had been assumed that the mechanisms, and hence the stoichiometries, would be the same. In this context, the evidence suggesting that the stoichiometry of H+/ATP (n above) varied depending on system was surprising. Values based on measure ATP/2e- ratios, and H+/2e- ratios had suggested that n was 3 for mitochondria, and 4 for chloroplasts, but these values were based on the assumption of integer stoichiometries. Although all the F1F0-type ATP-synthases likely had a common origin, both the assumption that the stoichiometries are the same, and that n is integer, are called into question by emerging structural data (see below).


Structure of the F1 ATP-ase

The structure of the soluble (F1) portion of the ATP synthase from beef heart mitochondria has been solved by X-ray crystallography. The pictures below are from Abrahams, J.P., Leslie, A.G., Lutter, R. and Walker, J.E. (1994) Structure at 2.8 Å resolution of F1-ATPase from bovine heart mitochondria



Mechanism of the F1 ATP-ase

The ATP synthase operates through a mechanism in which the three active sites undergo a change in binding affinity for the reactants of the ATP-ase reaction, ATP, ADP and phosphate, as originally predicted by Paul Boyer. The change in affinity accompanies a change in the position of the g-subunit relative to the a, b-ring, which involves a rotation of the one relative to the other. In the direction of ATP synthesis, the rotation is driven by a flux of H+ down the proton gradient, through a coupling between the g-subunit, and the c-subunit of FO. This rotation has now been demonstrated experimentally.


Respiratory control

The dependence of oxidative phosphorylation on ADP reveals an important general feature of this process: Respiration is tightly coupled to the synthesis of ATP. Not only is ATP synthesis absolutely dependent on continued electron flow from substrates to oxygen, but electron flow in normal mitochondria occurs only when ATP is being synthesized as well. This regulatory phenomenon, called respiratory control, makes biological sense, because it ensures that substrates will not be oxidized wastefully. Instead, their utilization is controlled by the physiological need for ATP.

In most aerobic cells the level of ATP exceeds that of ADP by 4- to 10-fold. Respiration depends on ADP as a substrate for phosphorylation. When ATP is consumed at high rates, accumulation of ADP stimulates respiration, with concomitant activation of ATP resynthesis. Conversely, in a relaxed and well-nourished cell, ATP accumulates at the expense of ADP, and the depletion of ADP limits the rate of both electron transport and its own phosphorylation to ATP. Thus, the energy-generating capacity of the cell is closely attuned to its energy demands.

Experimentally, respiratory control is demonstrated by following oxygen utilization in isolated mitochondria. In the absence of added substrate or ADP, oxygen uptake, caused by oxidation of endogenous substrates, is slow. Addition of an oxidizable substrate, such as glutamate or malate, has but a small effect on the respiration rate. If ADP is then added, however, oxygen uptake proceeds at an enhanced rate until all of the added ADP has been converted to ATP, and then oxygen uptake returns to the basal rate. This stimulation of respiration is stoichiometric; that is, addition of twice as much ADP causes twice the amount of oxygen uptake at the enhanced rate. If excess ADP is present instead of oxidizable substrate, the addition of substrate in limiting amounts will stimulate oxygen uptake until the substrate is exhausted.

Full-size image (52 K)

 Two mechanisms of the control of respiration and ATP synthesis in mitochondria according to the utilization of energy (ATP). The first mechanism of respiratory control is based on the proton motive force Δp across the inner mitochondrial membrane (grey). Activation of the ATP-synthase (blue) by ADP, taken up via the ATP/ADP carrier (margenta), decreases Δp which in consequence stimulates the three proton pumps of the respiratory chain (complexes I, III and IV). For simplicity, only complex IV (cytochrome c oxidase) and its substrate (cytochrome c) are shown in green and red, respectively. The second mechanism of respiratory control is based on the intramitochondrial ATP/ADP ratio. High ATP/ADP ratios inhibit cytochrome c oxidase activity allosterically. Uptake of ADP decreases the intramitochondrial ATP/ADP ratio accompanied by exchange of bound ATP by ADP at the matrix domain of subunit IV of cytochrome c oxidase, with subsequent stimulation of respiration

Maintenance of respiratory control depends on the structural integrity of the mitochondrion. Disruption of the organelle causes electron transport to become uncoupled from ATP synthesis. Under these conditions, oxygen uptake proceeds at high rates even in the absence of added ADP. ATP synthesis is inhibited, even though electrons are being passed along the respiratory chain and used to reduce O2 to water.

Uncoupling of respiration from phosphorylation can also be achieved chemically. Chemical uncouplers such as DNP or FCCP act by dissipating the proton gradient. Addition of an uncoupler to mitochondria stimulates oxygen utilization even in the absence of added ADP. No phosphorylation occurs under these conditions because there is no ADP to be phosphorylated.

The phenomenon of respiratory control is the subject of today's studio exercise. An oxygen electrode may be used to record [O2] in a closed vessel (diagram p. 804). Electron transfer, e.g., from NADH to O2, is monitored by recording the rate of disappearance of O2. At right is an idealized representation of an oxygen electrode recording while mitochondria respire in the presence of Pi, along with an electron donor (e.g., succinate, or a substrate of a reaction that will generate NADH). The dependence of respiration rate on availability of ADP, the substrate for the ATP Synthase, is called respiratory control. The respiratory control ratio is the ratio of slopes after and before ADP addition (b/a).

The P/O ratio is the moles of ADP added, divided by the moles of O consumed (based on c) while phosphorylating the added ADP.



Inhibitors of tissue respiration

Chemiosmosis can be disrupted by a variety of chemicals. In oxidative phosphorylation, some of these inhibitors are quite infamous:

Respiratory chain inhibitors.

The electron transport chain was determined by studying the effects of particular inhibitors.

Rotenone is a common insecticide that strongly inhibits the electron transport of complex I.

Rotenone is a natural product obtained from the roots of several species of plants. Tribes in certain parts of the world beat the roots of trees along riverbanks to release rotenone into the water which paralyzes fish and makes them easy prey.


Amytal is a barbiturate that inhibits the electron transport of complex I. Demerol is painkiller that also inhibits complex I. Amytal is a barbiturate drug that blocks electron transport from NADH to coenzyme Q. Amytal blocks electron transport at the same point as the insecticide rotenone.

Antibiotic. Induces apoptosis, which is not prevented by the presence of Bcl-2. Inhibits mitochondrial electron transport specifically between cytochromes b and c1. All three of these complex I inhibitors block the oxidation of the Fe-S clusters of complex I.

2-Thenoyltrifluoroacetone and carboxin specifically block electron transport in Complex II


Antimycin A is an antibiotic that inhibits electron transfer in complex III by blocking the transfer of electrons between Cyt bH and coenzyme Q bound at the QN site. Antibiotic. Induces apoptosis, which is not prevented by the presence of Bcl-2. Inhibits mitochondrial electron transport specifically between cytochromes b and c1.




Cyanide, azide and carbon monoxide all inhibit electron transport in Complex IV. The all inhibit electron transfer by binding tightly with the iron coordinated in Cyt a

  This complex oxidizes cytochrome c and also reduces O2 to H2O.    Remember that cytochromes have heme cofactors -- this is important in our discussion of cyanide and azide.    Cytochrome c is a soluble protein and also is a mobile carrier.  Other inhibitors of cytochrome c oxidase will not be discussed here, but are important biologically, such as sulfide, formate, and nitric oxide.

Azide and cyanide bind to the iron when the iron is in the ferric state. Carbon Monoxide binds to the iron when it is in the ferrous state. Cyanide and azide are potent inhibitors at this site which accounts for there acute toxicity. Carbon monoxide is toxic due to its affinity for the heme iron of hemoglobin. Animals carry many molecules of hemoglobin, therefore it takes a large quantity of carbon monoxide to die from carbon monoxide poisoning.


Carbon Monoxide

CO competes with oxygen for binding to the reduced form of cytochrome c oxidase.  Once bound to the cytochrome oxidase, oxygen cannot attach, and electron transport is stopped.  CO is a colorless, tasteless, non-irritating toxic gas.  When inhaled, the toxic gas enters the bloodstream, depriving the heart and brain of the oxygen necessary to function correctly. Sensing the body's need for more oxygen, the victim's heart rate increases to pump more blood to the body's organs. If a person continues to inhale CO, he or she faces the risk of breathing difficulty, cardiac trauma, brain damage, coma and even death.

Animals have relatively few molecules of Cyt a3. Consequently an exposure to a small quantity of azide or cyanide can be lethal. The toxicity of cyanide is solely from its ability to arrest electron transport. Cyanide

CN-, like azide, binds to the iron atom of oxidized cytochrome, preventing binding of oxygen.  Again, since cytochrome oxidase is inhibited, oxygen metabolism is prevented and thus so is energy generation.  Cyanide has long been known as a poison, sometimes used in warfare.  As mentioned above, lethal doses cause death in 15 minutes.


How does cyanide, azide or carbon monoxide cause death?

They all cause similar toxic repercussions (with the exception of Carbon Monoxide, which can bind to Hemoglobin causing your body to be unable to bind oxygen properly and causing you to suffocate);
However, these three also cause chemical suffocation. They bind a protein in our electron transport chain (cytochrome c oxidase). The electron transport chain is our body's best way to create energy in the form of ATP. Without it, we literally have next to no energy. The electron transport chain is the reason we have pain in our muscles when we work out--if our body doesn't have oxygen in those tissues and can't make energy, we make lactic acid, which causes our muscles to hurt/burn. Anyway, cyanide, azide and carbon monoxide bind this protein (cytochrome c oxidase) in the electron transport chain. Doing this causes all of the electrons to stop transferring and no energy to be made. This causes cell death. The first place to experience cell death from cyanide, azide and carbon monoxide is your central nervous system--which is made up of your brain and spinal cord.
So the first thing to die when someone dies of cyanide, azide or carbon monoxide poisoning?

The coupling between electron transport and oxidative phosphorylation depends on the impermeability of the inner mitochondrial membrane to H+translocation. The only way for protons to go from the intermembrane space to the matrix is through ATP synthase. Uncouplers uncouple electron transport from oxidative phosphorylation. They collapse the chemiosmotic gradient by dissipating protons across the inner mitochondrial membrane. All of the uncouplers shown to the left, collapse the pH gradient by binding a proton on the acidic side of the membrane, diffusing through the inner mitochondrial membrane and releasing the proton on the membranes alkaline side.

Uncouplers of oxidative phosphorylation stimulate the rate of electron flow but not

ATP synthesis.

(a)At relatively low levels of an uncoupling agent, P/O ratios drop somewhat, but the cell can compensate for this by increasing the rate of electron flow; ATP levels can be kept relatively normal. At high levels of uncoupler, P/O ratios approach zero and the cell cannot maintain ATP levels.

(b) As amounts of an uncoupler increase, the P/O ratio decreases and the body struggles to make sufficient ATP by oxidizing more fuel. The heat produced by this increased rate of oxidation raises the body temperature. The P/O ratio is affected as noted in (a).

(c) Increased activity of the respiratory chain in the presence of an uncoupler requires the degradation of additional energy stores (glycogen and fat). By oxidizing more fuel in an attempt to produce the same amount of ATP, the organism loses weight. If the P/O ratio nears zero, the lack of ATP will be lethal

2,4-Dinitrophenol, dicumarol and carbonyl cyanide-p-trifluorocarbonyl-cyanide methoxyphenyl hydrazone (FCCP) all have hydrophobic character making them soluble in the bilipid membrane. All of these decouplers also have dissociable protons allowing them to carry protons from the intermembrane space to the matrix which collapses the pH gradient. The potential energy of the proton gradient is lost as heat DNP is a chemical uncoupler of electron transport and oxidative phosphorylation.



DNP permeabilizes the inner mitochondrial membrane to protons, destroying the proton gradient and, in doing so, uncouples the electron transport system from the oxidative phosphorylation. In this situation, electrons continue to pass through the electron transport system and reduce oxygen to water, but ATP is not synthesized in the process. The compound, trifluorocarbonylcyanide phenylhydrazone (FCCP), is also an uncoupler.


The phenolic group of DNP is usually dissociated at intracellular pH. However, a DNP molecule that approaches the inner mitochondrial membrane from the outside becomes protonated (because the pH is lower there). Protonation increases the hydrophobicity of DNP, allowing it to diffuse into the membrane and, by mass action, to pass through. Once inside, the higher pH of the matric deprotonates the phenolic hydroxyl again. Thus, DNP has the effect of transporting H+  back into the matrix, bypassing the F0 proton channel and thereby preventing ATP synthesis.

The link between electron transport and ATP synthesis is below. (a) In the presence of excess phosphate and substrate and intact mitochondria, oxygen is consumed only when ADP is added. When all of the added ADP has been converted into ATP, electron transport stops and oxygen consumption ceases. (b) The addition of 2,4-dintrophenol uncouples electron transfer from ATP synthesis. The oxygen is completely consumed in the absence of ADP. Endogenous Uncouplers Enable Organisms to Generate Heat



The uncoupling of oxidative phosphorylation from electron transport generates heat. Hibernating animals and newborne animals (including human beings) contain brown adipose tissue. The adipose tissue is brown due to the high mitochondria content of the tissue. An endogenous protein called thermogenin uncouples ATP synthesis from electron transport by opening up a passive proton channel (UCP-1) through the inner mitochondrial membrane. The collapse of the pH gradient generates heat. An uncoupling protein (also called thermogenin) is produced in brown adipose tissue of newborn mammals and hibernating mammals .

This protein of the inner mitochondrial membrane functions as a H+ carrier. The uncoupling protein blocks development of a H+ electrochemical gradient, thereby stimulating respiration. The free energy change associated with respiration is dissipated as heat. This "non-shivering thermogenesis" is costly in terms of respiratory energy unavailable for ATP synthesis, but it provides valuable warming of the organism.

Valinomycin combines with K ions to form a complex that passes through the inner mito chondrial membrane. So, as a proton is translocated out by electron transfer, a K ion moves in, and the potential across the membrane is lost. This reduces the yield of ATP per mole of protons flowing through ATP synthase (FoF1). In other words, electron transfer and phosphorrylation become uncoupled. In response to the decreased efficiency of ATP synthesis, the rate of electron transfer increases markedly. This results in an increase in the H gradient, in oxygen consumption, and in the amount of heat released.

Valinomycin is a potent antibiotic which acts as a potassium (K+) ionophore. Induces K+ conductivity in cell membranes. Also active in vitro against Mycobacterium Tuberculosis, and as an apoptosis inducer.

Valinomycin is obtained from the cells of several Streptomyces strains, among which "S. tsusimaensis" and S. fulvissimus. It is a member of the group of natural neutral ionophores because it does not have a residual charge. It consists of enantiomers D- and L-valine (Val), D-hydroxyvaleric acid and L-lactic acid. Structures are alternately bound via amide and ester bridges. Valinomycin is highly selective for potassium ions over sodium ions within the cell membrane. It functions as a potassium-specific transporter and facilitates the movement of potassium ions through lipid membranes "down" an electrochemical potential gradient. The stability constant K for the potassium-valinomycin complex is 106 and for the sodium-valinomycin complex only 10. This difference is important for maintaining the selectivity of valinomycin for the transport of potassium ions (and not sodium ions) in biological systems.

Oligomycin is a natural antibiotic isolated from Streptomyces diastatochromogenes which inhibits mitochondrial H+-ATP synthase. It is primarily found to act as an inhibitor of mitochondrial respiration and swelling. This antibiotic is widely used as an inhibitor of oxidative phosphorylation.
Oligomycin inhibits the H+- ATP-synthase by binding to the Oligomycin sensitivity-conferring protein (OSCP) at the F(o) subunits 6 and 9  which are found in the stalk of the F1F0-ATPase complex. This binding blocks the proton conductance and inhibits the synthesis of mitochondrial ATP.3-4 Because of its activity, it can also be used to reduce the number of parameters (such as ER Ca2+ release, exocytotoxicity and apopotosis) which are affected by mitochondrial depolarization.

Oligomycin, at high concentrations may also inhibit the plasma membrane Na+-K+-ATPase. Interaction of Oligomycin with the Na+ occlusion site on the extracellular side of Na/K-ATPase, delays Na+ release to the extracellular side without inducing a conformational change. Although Oligomycin stimulated Na+ binding to Na+/K+-ATPase, it inhibited  Na+/Na+ exchange, and did not affect either Na+-dependent AD/-ATP exchange or K+-dependent phosphatase activity.6-16

Proton Motive Force Drives Transport The primary purpose of the proton gradient is togenerate ATP by oxidative phosphorylation. The potential energy of the gradient can also be used for active transport. The inner mitochondrial membrane is impermeable to charged molecules.


ATP-ADP Translocase There are two specific systems to transport ADP and Pi into the mitochondrial matrix. A specific transport protein ATP-ADP translocase enables ATP and ADP to transverse the inner mitochondrial membrane. The transport of ADP in and ATP out are coupled. ADP only enters the matrix if ATP exits or vice versa. ATP-ADP translocase has a single nucleotide binding site which binds ADP and ATP with equal affinity. Due to the negative electrostatic charge of the matrix, ATP-is bound on the N phase of the membrane because it has greater negative charge than ADP.

Pentachlorophenol (PCP) acts in a similar way to DNP. It was widely used as a biocide, especially in pallet board manufacture as a fungicide, but is now banned by the Biocidal Products Directive, because of its extreme toxicity and environmental persistence.



Ways of energy usage in the organism.

o                     1. From the smallest, single-celled organism to the biggest and most complex mammals--including people--all living things require energy for life. It's easy enough to understand that we and other animals eat. Things get a little more puzzling when we think about fungi, which absorb their food as organic molecules, from the surrounding environment. Where do those molecules come from? Furthermore, where does the food come from that we humans convert to energy? At the most basic level, all energy traces back to plants. Plants are the basis of all the world's food systems, and their unique ability to make organic materials from sunlight--called photosynthesis--is what sustains nearly every other life form on the planet.

o                     The powerhouse of energy production in all plants is called a chloroplast. More than a million of these handy devices occur in every quarter-inch of a leaf. They contain the pigment called chlorophyll that makes most leaves green--and drives photosynthesis. The reaction isn't all that complicated, as far as chemical reactions go. The chloroplasts take in carbon dioxide, sunlight and water. They release oxygen and a bit less water than they took in. The conversion of carbon dioxide to oxygen is one life-sustaining function that plants perform for Earth and all of its life. But plants do something equally as important when they keep a third product behind: glucose, the sugar that sustains the plants---and anything, in turn, that eats the plants.

o                     In cellular respiration, glucose is broken down by the removal of its hydrogen atoms. That process releases energy in the form of electrons, negatively charged particles that fuel all of a cell's other work in later reactions. So, plants make the glucose and everything down the line---from plant-eaters to the carnivores that eat them---break the glucose down again, and use its energy. That's the simple story. Of course, life is rarely so simple, and there are exceptions to every rule. Every so often, a new discovery comes along about living things that use a non-living substance other than sunlight to make energy--like ammonia, or even sulfur. These less-common organisms can harness electrons from chemical sources instead of the sun. More amazing life forms have the potential to be discovered at any time, anywhere on our planet---or beyond.


Cytochrome P450

The active site of cytochrome P450 contains a heme iron center. The iron is tethered to the P450 protein via a thiolate ligand derived from a cysteine residue. This cysteine and several flanking residues are highly conserved in known CYPs and have the formal PROSITE signature consensus pattern [FW] - [SGNH] - x - [GD] - {F} - [RKHPT] - {P} - C - [LIVMFAP] - [GAD].[7] Because of the vast variety of reactions catalyzed by CYPs, the activities and properties of the many CYPs differ in many aspects.