INVESTIGATION OF BIOLOGICAL OXIDATION, OXIDATIVE PHOSPHORYLATION AND ATP SYNTHESIS. INHIBITORS AND UNCOUPLERS OF OXIDATIVE PHOSPHORYLATION.

 

Bioenergetic

 

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.

The materials are generally combined with oxygen to release energy, although some can also be oxidized anaerobically by various organisms. The bonds holding the molecules of nutrients together and the bonds holding molecules of free oxygen together are all relatively weak compared with the chemical bonds holding carbon dioxide and water together. The utilization of these materials is a form of slow combustion. That is why the energy content of food can be estimated with a bomb calorimeter. The materials are oxidized slowly enough that the organisms do not actually produce fire. The oxidation releases energy because stronger bonds have been formed. This net energy may evolve as heat, or some of which may be used by the organism for other purposes, such as breaking other bonds to do chemistry.

Living organisms produce ATP from energy sources via oxidative phosphorylation. The terminal phosphate bonds of ATP are relatively weak compared with the stronger bonds formed when ATP is broken down to adenosine monophosphate and phosphate, dissolved in water. Here it is the energy of hydration that results in energy release. This hydrolysis of ATP is used as a battery to store energy in cells, for intermediate metabolism. Utilization of chemical energy from such molecular bond rearrangement powers biological processes in every biological organism.

 

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.

 

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.

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.

 

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.

 

Except for PP_i → 2 P_i, 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 + PP_i, to effectively supply the energy of hydrolysis of two high-energy bonds, with the hydrolysis of PP_i 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.

Fritz Albert Lipmann

Lipmann’s term “high-energy bond” and his symbol ~P (squiggle P) for a compound having a high phosphate group transfer potential are vivid, concise, and useful notations. In fact Lipmann’s squiggle did much to stimulate interest in bioenergetics.

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:

UMP, UDP, UTP, CMP, CDP, CTP, GMP, GDP, GTP.

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 mitochondrion’s 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 (PP_i) together or by bonding adenosine diphosphate (ADP) and one inorganic phosphate group (P_i) 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

 

 

 

•The bonds between the phosphate groups of ATP’s 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 + P_i              

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

ATP + H₂O → AMP + PP_i          

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

Hydrolysis of ATP

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

http://www.youtube.com/watch?v=_PgjsfY71AM&feature=related

http://www.youtube.com/watch?v=YndC0gS3t6M&feature=related

 

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.

 

oxidative phosphorylation

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 6.2.1.5) or a GDP-forming succinate-CoA ligase (G-SUCL, EC 6.2.1.4). 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

Redoxpotential

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.

Oxido-reductases

These enzymes catalyze oxidation-reduction reactions.

They are classified into five groups:

1-oxidases. 2-aerobic dehydrogenises.

3-anaerobic dehydrogenises.

4-hydroperoxidasesand

5-oxygenases.

 

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.

 

OXIDATION-REDUCTION OR REDOX REACTIONS

 

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

b)Isocitratedehydrogenase.

c)Malatedehydrogenase.

d)β-HydroxyacylCoAdehydrogenase.

e)β-Hydroxybutyrate dehydrogenase.

 

•NADP linked anaerobic dehydrogenasese.g.

a)Glucose-6-phosphate dehydrogenase.

b)Malicenzyme.c)Cytoplasmicisocitratedehydrogenase

 

•FAD linked anaerobic dehydrogenasese.g.

a)Succinatedehydrogenase.

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 (O•2). 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.

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

CytochromeP450

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:

•MicrosomalcytochromeP450.

 

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 1.6.5.3) (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.

Function

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.

 

 

 

 

 

 

Mechanism

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). ^[2]

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 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.

Ubiquinones

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.

History

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).

Types

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:

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:

Cytochromes	Combination
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	Plastoquinol—plastocyanin 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 F_O. 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.

 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 P_i, 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.

Chemiosmotic explanation of respiratory control: 

Electron transfer is obligatorily coupled to H⁺ ejection from the matrix. Whether this coupled reaction is spontaneous depends on the pH and electrical gradients.

*DG^o' = - nFDE^o' = -218 kJ/mol, for transfer of 2 e^- from NADH to O₂.

** For ejection of one H⁺ from the matrix:
DG = RT ln ([H⁺]_cytosol/[H⁺]_matrix) + F DY = 2.3 RT (pH_matrix - pH_cytosol) + F DY

In the absence of ADP, H⁺ cannot flow back to the matrix through F_o. The pH and electrical gradients (DpH & DY) are maximal. As respiration with outward H⁺ pumping proceeds, the free energy change for H⁺ ejection (positive DG) increases and approaches the magnitude of that for electron transfer (negative DG). When the coupled reaction becomes non-spontaneous, respiration stops. This is referred to as a static head. In fact there is usually a low rate of respiration in the absence of ADP, attributed to H⁺ leaks. Protons pumped out are carried by the uncoupler back into the mitochondrial matrix, preventing development of a pH or electrical gradient.

Inhibitors of tissue respiration

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

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.^1-2

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.

DNP

PCP

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. In general, the P450 catalytic cycle proceeds as follows:

1.     The substrate binds to the active site of the enzyme, in close proximity to the heme group, on the side opposite to the peptide chain. The bound substrate induces a change in the conformation of the active site, often displacing a water molecule from the distal axial coordination position of the heme iron,^[8] and sometimes changing the state of the heme iron from low-spin to high-spin.^[9] This gives rise to a change in the spectral properties of the enzyme, with an increase in absorbance at 390 nm and a decrease at 420 nm. This can be measured by difference spectrometry and is referred to as the "type I" difference spectrum (see inset graph in figure). Some substrates cause an opposite change in spectral properties, a "reverse type I" spectrum, by processes that are as yet unclear. Inhibitors and certain substrates that bind directly to the heme iron give rise to the type II difference spectrum, with a maximum at 430 nm and a minimum at 390 nm (see inset graph in figure). If no reducing equivalents are available, this complex may remain stable, allowing the degree of binding to be determined from absorbance measurements in vitro

2.     The change in the electronic state of the active site favors the transfer of an electron from NAD(P)H via cytochrome P450 reductase or another associated reductase^[11] This takes place by way of the electron transfer chain, as described above, reducing the ferric heme iron to the ferrous state.

3.     Molecular oxygen binds covalently to the distal axial coordination position of the heme iron. The cysteine ligand is a better electron donor than histidine, which is normally found in heme-containing proteins. As a consequence, the oxygen is activated to a greater extent than in other heme proteins. However, this sometimes allows the iron-oxygen bond to dissociate, causing the so-called "uncoupling reaction", which releases a reactive superoxide radical and interrupts the catalytic cycle.

4.      A second electron is transferred via the electron-transport system, from either cytochrome

  P450 reductase, ferredoxins, or cytochrome b5, reducing the dioxygen adduct to a negatively charged peroxo group. This is a short-lived intermediate state.

5.     The peroxo group formed in step 4 is rapidly protonated twice by local transfer from water or from surrounding amino-acid side-chains, releasing one water molecule, and forming a highly reactive species commonly referred to as P450. This highly reactive intermediate was not "seen in action" until 2010,^[12] although it had been studied theoretically for many years. P450 Compound 1 is most likely a iron(IV)oxo (or ferryl) species with an additional oxidizing equivalent delocalized over the porphyrin and thiolate ligands. Evidence for the alternative perferryl iron(V)-oxo ^[8] is lacking.

6.     Depending on the substrate and enzyme involved, P450 enzymes can catalyze any of a wide variety of reactions. A hypothetical hydroxylation is shown in this illustration. After the product has been released from the active site, the enzyme returns to its original state, with a water molecule returning to occupy the distal coordination position of the iron nucleus.

S: An alternative route for mono-oxygenation is via the "peroxide shunt": Interaction with single-oxygen donors such as peroxides and hypochlorites can lead directly to the formation of the iron-oxo intermediate, allowing the catalytic cycle to be completed without going through steps 2, 3, 4, and 5. A hypothetical peroxide "XOOH" is shown in the diagram.

C: If carbon monoxide (CO) binds to reduced P450, the catalytic cycle is interrupted. This reaction yields the classic CO difference spectrum with a maximum at 450 nm.

 

 

 

Free Radicals: Atoms contain a nucleus, and electrons move around the nucleus, usually in pairs. A free radical is any atom or molecule that contains one or more unpaired electrons.  The unpaired electrons alter the chemical reactivity of an atom or molecule, usually making it more reactive than the corresponding non-radical. However, the actual chemical reactivity of radicals varies enormously. 
           The hydrogen radical, the same as a hydrogen atom), which contains 1 proton and 1 electron (therefore unpaired), is the simplest free radical. Free-radical chain reactions are often initiated by removal of  from other molecules. A superscripted dot is used to denote free radicals. 

 

 

Free radicals and chain reaction:  Most molecules in the body are not radicals. Hence any reactive free radical generated is likely to react with a non-radical. When a free radical reacts with a non-radical, a free-radical chain reaction results and new radicals are formed. Figure 1 shows two important reactions of this type. Attack of reactive radicals on membranes or lipoproteins starts lipid peroxidation, which is particularly implicated in the development of atherosclerosis. If hydroxyl radicals are generated close to DNA, they can attack the purine and pyrimidine bases and cause mutations. For example, guanine is converted into 8-hydroxyguanine and other products.

Free radicals and diseases: Free radicals are capable of damage biomolecules, provoke immune response, activate oncogens, cause atherogenesis and enhance ageing process. However, in healthy conditions nature has endowed human body with enormous antioxidant potential. Subtle balance exists between free radical generation and antioxidant defence system to cope with oxidative stress by various enzymes and vitamins at cellular level which prevent the occurrence of disease. However, factors tilting the balance in favour of excess free radicals generation lead to widespread oxidative tissue damage and diseases. Therefore, trouble starts when there is an excess of free radicals and the defence mechanism lags behind. Overwhelming production of free radicals in response to exposure to toxic chemicals and ageing may necessitate judicious antioxidant supplement to help alleviate free radical mediated damage.

 

Free radicals with pollutants: Highly reactive molecules called free radicals can cause tissue damage by reacting with polyunsaturated fatty acids in cellular membranes, nucleotides in DNA, and critical sulfhydryl bonds in proteins. Free radicals can originate endogenously from normal metabolic reactions or exogenously as components of tobacco smoke and air pollutants and indirectly through the metabolism of certain solvents, drugs, and pesticides as well as through exposure to radiation.

There is some evidence that free radical damage contributes to the etiology of many chronic health problems such as emphysema, cardiovascular and inflammatory diseases, cataracts, and cancer. Defenses against free radical damage include tocopherol (vitamin E), ascorbic acid (vitamin C), beta-carotene, glutathione, uric acid, bilirubin, and several metalloenzymes including glutathione peroxidase (selenium), catalase (iron), and superoxide dismutase (copper, zinc, manganese) and proteins such as ceruloplasmin (copper). The extent of tissue damage is the result of the balance between the free radicals generated and the antioxidant protective defense system. Several dietary micronutrients contribute greatly to the protective system. Based on the growing interest in free radical biology and the lack of effective therapies for many of the chronic diseases, the usefulness of essential, safe nutrients in protecting against the adverse effects of oxidative injury warrants further st

 

WHAT CAN STOP A FREE RADICAL? (ANTIOXIDANTS): A free radical is stopped when the electron difference (gaining or losing an electron) is corrected.  Molecules that can correct the electron difference are called Antioxidants.  The process of damage by Free Radicals is called oxidation (think rust on metal or the browning of a cut apple), and the process that prevents it is anti-oxidation, and the molecules which do the prevention are called Antioxidants. Antioxidants are found in dark colored vegetables and fruit and in dietary supplements. The life of a free radical has three stages: the initiation stage, propagation stage, and finally the termination stage. Free radicals are terminated or neutralized by nutrients (antioxidants), enzymatic mechanisms, or by recombining with each other.