Investigation of biological oxidation, oxidative phosphorylation and ATP synthesis. Inhibitors and uncouplers of oxidative phosphorylation.
ATP and Other Nucleoside Triphosphates or: Bonds Rich in Energy
ATP is regarded as a universal source of energy occuring in all cell types. It is produced mainly during the oxidation of energy-rich (reduced) compounds processed in the respiratory chain and in photosynthesis.
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.)
ATP occurs usually as a magnesium or a manganese salt. For its hydrolysis, magnesium ions are necessary. Whenever it is spoken of ATP-degradation, it is always the hydrolysis of the terminal phosphate group(s) that is meant. The reactions are reversible:
ATP + H2O < > ADP + H3PO4 (= Pi)
ATP + H2O < > AMP + pyrophosphate (= PP)
ADP + H2O < > AMP + Pi
The phosphates are linked anhydrously, the innermost phosphate residue and the sugar residue are linked by an ester bond. Hydrolysis depends on the pH. The delta G° is -7.3 kcal/mol (ca -30.6 kJ/mol) at pH 7, i.e. at almost physiological conditions. It increases with a rising pH and is -10 kcal/mol (ca -42 kJ/mol) at pH
Since the delta G° for the breakdown of a pyrophosphate and for that of one phosphate residue are roughly the same, ATP, ADP and AMP can rather easily be converted into each other:
ATP + AMP < > 2 ADP
The delta G of the ATP breakdown is not very high compared to other phosphorylated compounds. Under this aspect, the term 'energy-rich linkage' seems irritating, but it has gained acceptance in biochemical literature as its hydrolysis is easily performed (with the help of the respective enzyme) and the energy is actually useable. The reason for the rather easily broken down linkage is in the electron accumulation at the terminal phosphate residues. Identical charges (here they are negative) repel each other and are in this case neutralized by hydrolysis.
In many cases, the terminal phosphate residue that is cleaved off from the ATP is not given away into solution as a free inorganic phosphate, but is transferred onto another molecule that becomes consequently phosphorylated. This process works also the other way round: a phosphorylated compound with a delta G° > -8 kcal/mol (-34 kJ/mol) can transfer its phosphate residue to ADP that as a consequence becomes ATP.
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.
Table of Contents
Adenosine triphosphate (ATP), the energy currency or coin of the cell pictured in Figfures 1 and 2, transfers energy from chemical bonds to endergonic (energy absorbing) reactions within the cell. Structurally, ATP consists of the adenine nucleotide (ribose sugar, adenine base, and phosphate group, PO4-2) plus two other phosphate groups.
Energy is stored in the covalent bonds between phosphates, with the greatest amount of energy (approximately 7 kcal/mole) in the bond between the second and third phosphate groups. This covalent bond is known as a pyrophosphate bond.
We can write the chemical reaction for the formation of ATP as:
a) in chemicalese: ADP + Pi + energy ----> ATP
b) in English: Adenosine diphosphate + inorganic Phosphate + energy produces Adenosine Triphosphate
The chemical formula for the expenditure/release of ATP energy can be written as:
a) in chemicalese: ATP ----> ADP + energy + Pi
b) in English Adenosine Triphosphate produces Adenosine diphosphate + energy + inorganic Phosphate
An analogy between ATP and rechargeable batteries is appropriate. The batteries are used, giving up their potential energy until it has all been converted into kinetic energy and heat/unusable energy. Recharged batteries (into which energy has been put) can be used only after the input of additional energy. Thus, ATP is the higher energy form (the recharged battery) while ADP is the lower energy form (the used battery). When the terminal (third) phosphate is cut loose, ATP becomes ADP (Adenosine diphosphate; di= two), and the stored energy is released for some biological process to utilize. The input of additional energy (plus a phosphate group) "recharges" ADP into ATP (as in my analogy the spent batteries are recharged by the input of additional energy).
Two processes convert ADP into ATP: 1) substrate-level phosphorylation; and 2) chemiosmosis. Substrate-level phosphorylation occurs in the cytoplasm when an enzyme attaches a third phosphate to the ADP (both ADP and the phosphates are the substrates on which the enzyme acts). This is illustrated in Figure 3.
Figure 3. Enzymes and the formation of NADH and ATP. Images from Purves et al., Life: The Science of Biology, 4th Edition, by Sinauer Associates (www.sinauer.com) and WH Freeman (www.whfreeman.com), used with permission.
shown in Figure 4, involves more than the single enzyme of substrate-level
phosphorylation. Enzymes in chemiosmotic synthesis are arranged in an electron transport chain
that is embedded in a membrane. In eukaryotes this membrane is in either the chloroplast
According to the chemiosmosis hypothesis proposed by Peter Mitchell in
During chemiosmosis in eukaryotes, H+ ions are pumped across an organelle membrane by membrane "pump proteins" into a confined space (bounded by membranes) that contains numerous hydrogen ions. This is shown in Figure 4 and 5. The energy for the pumping comes from the coupled oxidation-reduction reactions in the electron transport chain. Electrons are passed from one membrane-bound enzyme to another, losing some energy with each tansfer (as per the second law of thermodynamics). This "lost" energy allows for the pumping of hydrogen ions against the concentration gradient (there are fewer hydrogen ions outside the confined space than there are inside the confined space). The confined hydrogens cannot pass back through the membrane. Their only exit is through the ATP synthesizing enzyme that is located in the confining membrane. As the hydrogen passes through the ATP synthesizing enzyme, energy from the enzyme is used to attach a third phosphate to ADP, converting it to ATP.
Usually the terminal phosphate is not simply removed, but instead is attached to another molecule. This process is known as phosphorylation.
W + ATP -----> W~P + ADP where W is any compound, for example:
glucose + ATP -----> glucose~P + ADP
Glucose can be converted into Glucose-6-phosphate by the addition of the phosphate group from ATP.
· ATP is composed of ribose, a five-carbon sugar, three phosphate groups, and adenine , a nitrogen-containing compound (also known as a nitrogenous base). What class of organic macromolecules is composed of monomers similar to ATP?
· ATP directly or indirectly delivers energy to almost all metabolic pathways. Explain the functioning of the ATP/ADP cycle.
· Adding a phosphate to a molecule is called phosphorylation. What two methods do cells use to phosphorylate ADP into ATP?
ATP: The Perfect Energy Currency for the Cell
The major energy currency molecule of the cell, ATP, is evaluated in the context of creationism. This complex molecule is critical for all life from the simplest to the most complex. It is only one of millions of enormously intricate nanomachines that needs to have been designed in order for life to exist on earth. This motor is an excellent example of irreducible complexity because it is necessary in its entirety in order for even the simplest form of life to survive.
In order to function, every machine requires specific parts such as screws, springs, cams, gears, and pulleys. Likewise, all biological machines must have many well-engineered parts to work. Examples include units called organs such as the liver, kidney, and heart. These complex life units are made from still smaller parts called cells which in turn are constructed from yet smaller machines known as organelles. Cell organelles include mitochondria, Golgi complexes, microtubules, and centrioles. Even below this level are other parts so small that they are formally classified as macromolecules (large molecules).
Fig. 1. Views of ATP and related structures.
A critically important macromolecule—arguably “second in importance only to DNA”—is ATP. ATP is a complex nanomachine that serves as the primary energy currency of the cell (Trefil, 1992, p.93). A nanomachine is a complex precision microscopic-sized machine that fits the standard definition of a machine. ATP is the “most widely distributed high-energy compound within the human body” (Ritter, 1996, p. 301). This ubiquitous molecule is “used to build complex molecules, contract muscles, generate electricity in nerves, and light fireflies. All fuel sources of Nature, all foodstuffs of living things, produce ATP, which in turn powers virtually every activity of the cell and organism. Imagine the metabolic confusion if this were not so: Each of the diverse foodstuffs would generate different energy currencies and each of the great variety of cellular functions would have to trade in its unique currency” (Kornberg, 1989, p. 62).
ATP is an abbreviation for adenosine triphosphate, a complex molecule that contains the nucleoside adenosine and a tail consisting of three phosphates. (See Figure 1 for a simple structural formula and a space filled model of ATP.) As far as known, all organisms from the simplest bacteria to humans use ATP as their primary energy currency. The energy level it carries is just the right amount for most biological reactions. Nutrients contain energy in low-energy covalent bonds which are not very useful to do most of kinds of work in the cells.
These low energy bonds must be translated to high energy bonds, and this is a role of ATP. A steady supply of ATP is so critical that a poison which attacks any of the proteins used in ATP production kills the organism in minutes. Certain cyanide compounds, for example, are poisonous because they bind to the copper atom in cytochrome oxidase. This binding blocks the electron transport system in the mitochondria where ATP manufacture occurs (Goodsell, 1996, p.74).
How ATP Transfers Energy
Energy is usually liberated from the ATP molecule to do work in the cell by a reaction that removes one of the phosphate-oxygen groups, leaving adenosine diphosphate (ADP). When the ATP converts to ADP, the ATP is said to be spent. Then the ADP is usually immediately recycled in the mitochondria where it is recharged and comes out again as ATP. In the words of Trefil (1992, p. 93) “hooking and unhooking that last phosphate [on ATP] is what keeps the whole world operating.”
The enormous amount of activity that occurs inside each of the approximately one hundred trillion human cells is shown by the fact that at any instant each cell contains about one billion ATP molecules. This amount is sufficient for that cell’s needs for only a few minutes and must be rapidly recycled. Given a hundred trillion cells in the average male, about 1023 or one sextillion ATP molecules normally exist in the body. For each ATP “the terminal phosphate is added and removed 3 times each minute” (Kornberg, 1989, p. 65).
The total human body content of ATP is only about
The Structure of ATP
ATP contains the purine base adenine and the sugar ribose which together form the nucleoside adenosine. The basic building blocks used to construct ATP are carbon, hydrogen, nitrogen, oxygen, and phosphorus which are assembled in a complex that contains the number of subatomic parts equivalent to over 500 hydrogen atoms. One phosphate ester bond and two phosphate anhydride bonds hold the three phosphates (PO4) and the ribose together. The construction also contains a b-N glycoside bond holding the ribose and the adenine together.
Fig. 2. The two-dimensional stick model of the adenosine phosphate family of molecules, showing the atom and bond arrangement.
Phosphates are well-known high-energy molecules, meaning that comparatively high levels of energy are released when the phosphate groups are removed. Actually, the high energy content is not the result of simply the phosphate bond but the total interaction of all the atoms within the ATP molecule.
Because the amount of energy released when the phosphate bond is broken is very close to that needed by the typical biological reaction, little energy is wasted. Generally, ATP is connected to another reaction—a process called coupling which means the two reactions occur at the same time and at the same place, usually utilizing the same enzyme complex. Release of phosphate from ATP is exothermic (a reaction that gives off heat) and the reaction it is connected to is endothermic (requires energy input in order to occur). The terminal phosphate group is then transferred by hydrolysis to another compound, a process called phosphorylation, producing ADP, phosphate (Pi) and energy.
The self-regulation system of ATP has been described as follows:
The high-energy bonds of ATP are actually rather unstable bonds. Because they are unstable, the energy of ATP is readily released when ATP is hydrolyzed in cellular reactions. Note that ATP is an energy-coupling agent and not a fuel. It is not a storehouse of energy set aside for some future need. Rather it is produced by one set of reactions and is almost immediately consumed by another. ATP is formed as it is needed, primarily by oxidative processes in the mitochondria. Oxygen is not consumed unless ADP and a phosphate molecule are available, and these do not become available until ATP is hydrolyzed by some energy-consuming process. Energy metabolism is therefore mostly self-regulating (Hickman, Roberts, and Larson, 1997, p.43). [Italics mine]
ATP is not excessively unstable, but it is designed so that its hydrolysis is slow in the absence of a catalyst. This insures that its stored energy is “released only in the presence of the appropriate enzyme” (McMurry and Castellion, 1996, p. 601).
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 (Hoagland and Dodson, 1995, p.104). 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.
Fig. 3. An outline of the ATP-synthase macromolecule showing its subunits and nanomachine traits. ATP-synthase converts ADP into ATP, a process called charging. Shown behind ATP-synthase is the membrane in which the ATP-synthase is mounted. For the ATP that is charged in the mitochondria, ATP-synthase is located in the inner membrane.
The mitochondrion itself functions to produce an electrical chemical gradient—somewhat like a battery—by accumulating hydrogen ions in the space between the inner and outer membrane. This energy comes from the estimated 10,000 enzyme chains in the membranous sacks on the mitochondrial walls. Most of the food energy for most organisms is produced by the electron transport chain. Cellular oxidation in the Krebs cycle causes an electron build-up that is used to push H+ ions outward across the inner mitochondrial membrane (Hickman et al., 1997, p. 71).
As the charge builds up, it provides an electrical potential that releases its energy by causing a flow of hydrogen ions across the inner membrane into the inner chamber. The energy causes an enzyme to be attached to ADP which catalyzes the addition of a third phosphorus to form ATP. Plants can also produce ATP in this manner in their mitochondria but plants can also produce ATP by using the energy of sunlight in chloroplasts as discussed later. In the case of eukaryotic animals the energy comes from food which is converted to pyruvate and then to acetyl coenzyme A (acetyl CoA). Acetyl CoA then enters the Krebs cycle which releases energy that results in the conversion of ADP back into ATP.
How does this potential difference serve to reattach the phosphates on ADP molecules? The more protons there are in an area, the more they repel each other. When the repulsion reaches a certain level, the hydrogens ions are forced out of a revolving-door-like structure mounted on the inner mitochondria membrane called ATP synthase complexes. This enzyme functions to reattach the phosphates to the ADP molecules, again forming ATP.
The ATP synthase revolving door resembles a molecular water wheel that harnesses the flow of hydrogen ions in order to build ATP molecules. Each revolution of the wheel requires the energy of about nine hydrogen ions returning into the mitochondrial inner chamber (Goodsell, 1996, p.74). Located on the ATP synthase are three active sites, each of which converts ADP to ATP with every turn of the wheel. Under maximum conditions, the ATP synthase wheel turns at a rate of up to 200 revolutions per second, producing 600 ATPs during that second.
ATP is used in conjunction with enzymes to cause certain molecules to bond together. The correct molecule first docks in the active site of the enzyme along with an ATP molecule. The enzyme then catalyzes the transfer of one of the ATP phosphates to the molecule, thereby transferring to that molecule the energy stored in the ATP molecule. Next a second molecule docks nearby at a second active site on the enzyme. The phosphate is then transferred to it, providing the energy needed to bond the two molecules now attached to the enzyme. Once they are bonded, the new molecule is released. This operation is similar to using a mechanical jig to properly position two pieces of metal which are then welded together. Once welded, they are released as a unit and the process then can begin again.
A Double Energy Packet
Although ATP contains the amount of energy necessary for most reactions, at times more energy is required. The solution is for ATP to release two phosphates instead of one, producing an adenosine monophosphate (AMP) plus a chain of two phosphates called a pyrophosphate. How adenosine monophosphate is built up into ATP again illustrates the precision and the complexity of the cell energy system. The enzymes used in glycolysis, the citric acid cycle, and the electron transport system, are all so precise that they will replace only a single phosphate. They cannot add two new phosphates to an AMP molecule to form ATP.
The solution is an intricate enzyme called adenylate kinase which transfers a single phosphate from an ATP to the AMP, producing two ADP molecules. The two ADP molecules can then enter the normal Krebs cycle designed to convert ADP into ATP. Adenylate kinase requires an atom of magnesium—and this is one of the reasons why sufficient dietary magnesium is important.
Adenylate kinase is a highly organized but compact enzyme with its active site located deep within the molecule. The deep active site is required because the reactions it catalyzes are sensitive to water. If water molecules lodged between the ATP and the AMP, then the phosphate might break ATP into ADP and a free phosphate instead of transferring a phosphate from ATP to AMP to form ADP.
To prevent this, adenylate kinase is designed so that the active site is at the end of a channel deep in the structure which closes around AMP and ATP, shielding the reaction from water. Many other enzymes that use ATP rely on this system to shelter their active site to prevent inappropriate reactions from occurring. This system ensures that the only waste that occurs is the normal wear, tear, repair, and replacement of the cell’s organelles.
Pyrophosphates and pyrophosphoric acid, both inorganic forms of phosphorus, must also be broken down so they can be recycled. This phosphate breakdown is accomplished by the inorganic enzyme pyrophosphatase which splits the pyrophosphate to form two free phosphates that can be used to charge ATP (Goodsell, 1996, p.79). This system is so amazingly efficient that it produces virtually no waste, which is astounding considering its enormously detailed structure. Goodsell (1996, p. 79) adds that “our energy-producing machinery is designed for the production of ATP: quickly, efficiently, and in large quantity.”
The main energy carrier the body uses is ATP, but other energized nucleotides are also utilized such as thymine, guanine, uracil, and cytosine for making RNA and DNA. The Krebs cycle charges only ADP, but the energy contained in ATP can be transferred to one of the other nucleosides by means of an enzyme called nucleoside diphosphate kinase. This enzyme transfers the phosphate from a nucleoside triphosphate, commonly ATP, to a nucleoside diphosphate such as guanosine diphosphate (GDP) to form guanosine triphosphate (GTP).
The nucleoside diphosphate kinase works by one of its six active sites binding nucleoside triphosphate and releasing the phosphate which is bonded to a histidine. Then the nucleoside triphosphate, which is now a diphosphate, is released, and a different nucleoside diphosphate binds to the same site—and as a result the phosphate that is bonded to the enzyme is transferred, forming a new triphosphate. Scores of other enzymes exist in order for ATP to transfer its energy to the various places where it is needed. Each enzyme must be specifically designed to carry out its unique function, and most of these enzymes are critical for health and life.
The body does contain some flexibility, and sometimes life is possible when one of these enzymes is defective—but the person is often handicapped. Also, back-up mechanisms sometimes exist so that the body can achieve the same goals through an alternative biochemical route. These few simple examples eloquently illustrate the concept of over-design built into the body. They also prove the enormous complexity of the body and its biochemistry.
The Message of the Molecule
Without ATP, life as we understand it could not exist. It is a perfectly-designed, intricate molecule that serves a critical role in providing the proper size energy packet for scores of thousands of classes of reactions that occur in all forms of life. Even viruses rely on an ATP molecule identical to that used in humans. The ATP energy system is quick, highly efficient, produces a rapid turnover of ATP, and can rapidly respond to energy demand changes (Goodsell, 1996, p.79).
Furthermore, the ATP molecule is so enormously intricate that we are just now beginning to understand how it works. Each ATP molecule is over 500 atomic mass units (500 AMUs). In manufacturing terms, the ATP molecule is a machine with a level of organization on the order of a research microscope or a standard television (Darnell, Lodish, and Baltimore, 1996).
Among the questions evolutionists must answer include the following, “How did life exist before ATP?” “How could life survive without ATP since no form of life we know of today can do that?” and “How could ATP evolve and where are the many transitional forms required to evolve the complex ATP molecule?” No feasible candidates exist and none can exist because only a perfect ATP molecule can properly carry out its role in the cell.
In addition, a potential ATP candidate molecule would not be selected for by evolution until it was functional and life could not exist without ATP or a similar molecule that would have the same function. ATP is an example of a molecule that displays irreducible complexity which cannot be simplified and still function (Behe, 1996). ATP could have been created only as a unit to function immediately in life and the same is true of the other intricate energy molecules used in life such as GTP.
Although other energy molecules can be used for certain cell functions, none can even come close to satisfactorily replacing all the many functions of ATP. Over 100,000 other detailed molecules like ATP have also been designed to enable humans to live, and all the same problems related to their origin exist for them all. Many macromolecules that have greater detail than ATP exist, as do a few that are less highly organized, and in order for life to exist all of them must work together as a unit.
The Contrast between Prokaryotic and Eukaryotic ATP Production An enormous gap exists between prokaryote (bacteria and cyanobacteria) cells and eukaryote (protists, plants and animals) type of cells:
...prokaryotes and eukaryotes are profoundly different from each other and clearly represent a marked dichotomy in the evolution of life. . . The organizational complexity of the eukaryotes is so much greater than that of the prokaryotes that it is difficult to visualize how a eukaryote could have arisen from any known prokaryote (Hickman et al., 1997, p. 39).
Some of the differences are that prokaryotes lack organelles, a cytoskeleton, and most of the other structures present in eukaryotic cells. Consequently, the functions of most organelles and other ultrastructure cell parts must be performed in bacteria by the cell membrane and its infoldings called mesosomes.
The Four Major Methods of Producing ATP
A crucial difference between prokaryotes and eukaryotes is the means they use to produce ATP. All life produces ATP by three basic chemical methods only: oxidative phosphorylation, photophosphorylation, and substrate-level phosphorylation (Lim, 1998, p. 149). In prokaryotes ATP is produced both in the cell wall and in the cytosol by glycolysis. In eukaryotes most ATP is produced in chloroplasts (for plants), or in mitochondria (for both plants and animals). No means of producing ATP exists that is intermediate between these four basic methods and no transitional forms have ever been found that bridge the gap between these four different forms of ATP production. The machinery required to manufacture ATP is so intricate that viruses are not able to make their own ATP. They require cells to manufacture it and viruses have no source of energy apart from cells.
In prokaryotes the cell membrane takes care of not only the cell’s energy-conversion needs, but also nutrient processing, synthesizing of structural macromolecules, and secretion of the many enzymes needed for life (Talaro and Talaro, 1993, p. 77). The cell membrane must for this reason be compared with the entire eukaryote cell ultrastructure which performs these many functions. No simple means of producing ATP is known and prokaryotes are not by any means simple. They contain over 5,000 different kinds of molecules and can use sunlight, organic compounds such as carbohydrates, and inorganic compounds as sources of energy to manufacture ATP.
Another example of the cell membrane in prokaryotes assuming a function of the eukaryotic cell ultrastructure is as follows: Their DNA is physically attached to the bacterial cell membrane and DNA replication may be initiated by changes in the membrane. These membrane changes are in turn related to the bacterium’s growth. Further, the mesosome appears to guide the duplicated chromatin bodies into the two daughter cells during cell division (Talaro and Talaro, 1993).
In eukaryotes the mitochondria produce most of the cell’s ATP (anaerobic glycolysis also produces some) and in plants the chloroplasts can also service this function. The mitochondria produce ATP in their internal membrane system called the cristae. Since bacteria lack mitochondria, as well as an internal membrane system, they must produce ATP in their cell membrane which they do by two basic steps. The bacterial cell membrane contains a unique structure designed to produce ATP and no comparable structure has been found in any eukaryotic cell (Jensen, Wright, and Robinson, 1997).
In bacteria, the ATPase and the electron transport chain are located inside the cytoplasmic membrane between the hydrophobic tails of the phospholipid membrane inner and outer walls. Breakdown of sugar and other food causes the positively charged protons on the outside of the membrane to accumulate to a much higher concentration than they are on the membrane inside. This creates an excess positive charge on the outside of the membrane and a relatively negative charge on the inside.
The result of this charge difference
is a dissociation of H2O molecules into H+ and
In some bacteria such as Halobacterium this system is modified by use of bacteriorhodopsin, a protein similar to the sensory pigment rhodopsin used in the vertebrate retina (Lim, 1998, p. 166). Illumination causes the pigment to absorb light energy, temporarily changing rhodopsin from a trans to a cis form. The trans to cis conversion causes deprotonation and the transfer of protons across the plasma membrane to the periplasm.
The proton gradient that results is used to drive ATP synthesis by use of the ATPase complex. This modification allows bacteria to live in low oxygen but rich light regions. This anaerobic ATP manufacturing system, which is unique to prokaryotes, uses a chemical compound other than oxygen as a terminal electron acceptor (Lim, 1998, p. 168). The location of the ATP producing system is only one of many major contrasts that exist between bacterial cell membranes and mitochondria.
Chloroplasts are double membraned ATP-producing organelles found only in plants. Inside their outer membrane is a set of thin membranes organized into flattened sacs stacked up like coins called thylakoids (Greek thylac or sack, and oid meaning like). The disks contain chlorophyll pigments that absorb solar energy which is the ultimate source of energy for all the plant’s needs including manufacturing carbohydrates from carbon dioxide and water (Mader, 1996, p. 75). The chloroplasts first convert the solar energy into ATP stored energy, which is then used to manufacture storage carbohydrates which can be converted back into ATP when energy is needed.
The chloroplasts also possess an electron transport system for producing ATP. The electrons that enter the system are taken from water. During photosynthesis, carbon dioxide is reduced to a carbohydrate by energy obtained from ATP (Mader, 1996, p. 12). Photosynthesizing bacteria (cyanobacteria) use yet another system. Cyanobacteria do not manufacture chloroplasts but use chlorophyll bound to cytoplasmic thylakoids. Once again plausible transitional forms have never been found that can link this form of ATP production to the chloroplast photosynthesis system.
The two most common evolutionary theories of the origin of the mitochondria-chloroplast ATP production system are 1) endosymbiosis of mitochondria and chloroplasts from the bacterial membrane system and 2) the gradual evolution of the prokaryote cell membrane system of ATP production into the mitochondria and chloroplast systems. Believers in endosymbiosis teach that mitochondria were once free-living bacteria, and that “early in evolution ancestral eukaryotic cells simply ate their future partners” (Vogel, 1998, p. 1633). Both the gradual conversion and endosymbiosis theory require many transitional forms, each new one which must provide the animal with a competitive advantage compared with the unaltered animals.
The many contrasts between the prokaryotic and eukaryotic means of producing ATP, some of which were noted above, are strong evidence against the endosymbiosis theory. No intermediates to bridge these two systems has ever been found and arguments put forth in the theory’s support are all highly speculative. These and other problems have recently become more evident as a result of recent major challenges to the standard endosymbiosis theory. The standard theory has recently been under attack from several fronts, and some researchers are now arguing for a new theory:
Scientists pondering how the first complex cell came
together say the new idea could solve some nagging problems with the prevailing
theory... “[the new theory is]... elegantly argued,” says Michael Gray of
In this brief review we have examined only one cell macromolecule, ATP, and the intricate mechanisms which produce it. We have also looked at the detailed supporting mechanism which allows the ATP molecule to function. ATP is only one of hundreds of thousands of essential molecules, each one that has a story. As each of those stories is told, they will stand as a tribute to both the genius and the enormously complex design of the natural world. All the books in the largest library in the world may not be able to contain the information needed to understand and construct the estimated 100,000 complex macromolecule machines used in humans. Much progress has been made in understanding the structure and function of organic macromolecules and some of the simpler ones are now being manufactured by pharmaceutical firms.
Now that scientists understand how some of these highly organized molecules function and why they are required for life, their origin must be explained. We know only four basic methods of producing ATP: in bacterial cell walls, in the cytoplasm by photosynthesis, in chloroplasts, and in mitochondria. No transitional forms exist to bridge these four methods by evolution. According to the concept of irreducible complexity, these ATP producing machines must have been manufactured as functioning units and they could not have evolved by Darwinism mechanisms. Anything less than an entire ATP molecule will not function and a manufacturing plant which is less then complete cannot produce a functioning ATP. Some believe that the field of biochemistry which has achieved this understanding has already falsified the Darwinian world view (Behe, 1996).
A substance called adenosine triphosphate (ATP) links most cellular exergonic (def) and endergonic (def) chemical reactions. To obtain energy to do cellular work, organisms take energy-rich compounds such as glucose into the cell and enzymatically break them down to release their potential energy. Therefore, the organism needs a way to trap some of that released energy and store the energy in a form that can be utilized by the cell to do cellular work. Principally, energy is trapped and stored in the form of adenosine triphosphate (def) or ATP.
A tremendous amount of ATP is needed for normal cellular growth. For
example,a human at rest uses about
To trap energy released from exergonic catabolic chemical reactions (def), the cell uses some of that released energy to attach an inorganic phosphate group on to adenosine diphosphate (ADP) to make adenosine triphosphate (ATP). Thus, energy is trapped and stored in what are known as high-energy phosphate bonds. To obtain energy to do cellular work during endergonic anabolic chemical reactions (def), the organism enzymatically removes the third phosphate from ATP thus releasing the stored energy and forming ADP and inorganic phosphate once again (see Fig. 1).
Depending on the type of organism, cells transfer energy and generate ATP by photophosphorylation, by substrate-level phosphorylation, and/or by oxidative phosphorylation. (Phosphorylation (def) refers to the attachment of a phosphate group to a molecule.)
Oxidative phosphorylation in eukaryotes occurs exclusively in their mitochondria.
As previously discussed, mitochondria convert pyruvate into carbon dioxide and water via the Krebs cycle. This produces NADH and a little ATP. The NADH, which would otherwise accumulate until there was no NAD left, is re-oxidised by the oxidative phosphorylation respiratory chain to regenerate the NAD, and (as a fabulous bonus), generate loads of ATP too, because this oxidation is coupled to the production of a proton gradient across their inner membrane, and these protons flow down their gradient via FOF1-ATPase (ATP synthase), making ATP.
Mitochondria are believed to be the product of an endosymbiosis 2500 MYA. They probably originated from an intracellular proteobacterial parasite of proto-eukaryotic cells (something like Bdellovibrio). Chloroplasts derived from a similar symbiosis with cyanobacteria (closely related to Prochloron).
Since oxidative phosphorylation is much more efficient (30 vs. 2 ATP per glucose) than anaerobic respiration, and photosynthesis allows growth in the absence of exogenous carbon and reducing agents, what may have begun as a parasitic or predatory relationship between the Ur-eukaryote and its bacterial passengers, developed into a mutualism. The mitochondrial symbiosis were probably a one-off, but chloroplasts may not have been. Dinoflagellates even have secondary endosymbionts (like Russian matryoshka dolls): a chloroplast within an alga within another alga. There is a great deal of evidence for endosymbiosis these days (the theory was once considered very unlikely):
· Inner membrane = Remains of bacterial plasmalemma.
· Outer membrane = Remains of food vacuole.
· Cristae = Like mesosome infoldings.
· Circular DNA lacking histones in matrix = Remains of bacterial chromosome.
· Small ribosomes = Like bacterial 70S ribosomes (eukaryotes have 80S).
The symbiosis has gone far beyond a simple ingestion. The human mtDNA (mitochondrial DNA) genome contains just 37 genes. These are mostly tRNAs, with some of the proteins of oxidative phosphorylation:
· 7/27 of Complex I
· 0/4 of Complex II
· 1/9 of Complex III
· 3/13 of Complex IV
· 2/12 of Complex V
The rest is now nuclear encoded and imported via the TOM/TIM transport system, showing that over evolutionary time, the genes of the mitochondria have either been lost, or (when essential) transferred to the nuclear genome.
The job of mitochondria is to convert pyruvate to ATP and carbon dioxide. This is achieved by the interaction of NADH, and one Krebs cycle intermediate (succinate) with the inner mitochondrial membrane. This membrane contains five huge protein complexes, which serve to remove electrons from NADH, regenerating NAD, and in-so-doing, to generate a proton gradient across the membrane than may be used to drive ATP synthesis.
The five complexes are named I, II, III, IV and V. We shall discuss them in turn:
Electrons from the oxidation of NADH pass through flavin,
Complex I is NADH dehydrogenase. It removes two electron from NADH, and transfers them to ubiquinone in the mitochondrial membrane. As the two electrons pass through various flavin (FMN), iron-sulfur (FeS) and quinone (UQ) centres, four protons are pumped across complex I into the inter-membrane space (per NADH). When the electrons are deposited onto UQ (ubiquinone), the UQ takes up a further two protons from the matrix side, to form ubiquinol (UQH2) (these are excluded from the pump-count for this complex). It produces 1 UQH2, per NADH oxidised. The ultimate source of the NADH and the electrons is the oxidation of ketoglutarate, malate etc in the Krebs cycle.
The ubiquinol formed feeds into a UQ 'pool' inside the membrane, and diffuses to complex III.
Succinate is oxidised to generate fumarate, and UQH2 from UQ.
Complex II is also called succinate dehydrogenase, and is the only membrane bound enzyme of the Krebs cycle. The dehydrogenation of succinate has too small a ∆G for any H+ pumping, so this complex only generates 1 UQH2 per succinate oxidised, and pumps no protons. It gets its electrons from the oxidation of succinate only, and feeds them via flavin (FAD) and an iron-sulfur cluster into the UQH2 pool.
Ubiquinone (UQ) ferries electrons from complexes I and II to complex III. Note the long hydrophobic chain: UQ/UQH2 can migrate actually dissolved within the membrane.
Partial reduction of UQ generates ubisemiquinone radicals (UQH·), which are very dangerous and must be rapidly reduced to UQH2.
Complexes I and II both feed into a pool of ubiquinol (UQH2) actually inside the inner mitochondrial membrane (dissolved in the fatty acid tails).
The reason semiubiquinone is dangerous is that is can generate superoxide radicals, which are hugely oxidising free-radicals.
UQH· + O2 → UQ + H+ + O2−·
Superoxide will dismutate to hydrogen peroxide.
2O2−· + 2H+ → O2 + H2O2
Hydrogen peroxide will undergo Fenton reaction with haem iron to produce hydroxyl radicals which are lethally destructive.
Fe2+ + H2O2 → Fe3+ + OH− + OH·
Mitochondria therefore contain superoxide dismutase and glutathione (GSH) peroxidase to cope with these agents of oxidative stress.
2GSH + H2O2 → GSSG + 2H2O
This is also called cytochrome reductase (or oxidoreductase). It pumps 4 H+ per UQH2 (including the two attached by complex I or II to UQ), and produces 2 cyt-cRED (reduced cytochrome-c) per UQH2 oxidised. The iron in the haem groups of b and c cytochromes goes from Fe3+ to Fe2+. The complex manages to pump 4 protons by running a nasty bit of biochemistry called the Q-cycle, which delivers the two electrons from one UQH2 to two cyt-c molecules, which only carry one electron each.
'Q-cycle' is a preposterously complicated way of transferring electrons from
the two-electron-carrying UQH2 to the single-electron carrying
cytochrome-c (cyt-c). A UQH2 gives up its protons to the IMS. One of
its electrons is carried through
One electron from UQH2 is used to reduce cyt-c, the other is used to half-reduce a UQ in the membrane to semiquinone. This is accompanied by the release of two protons per UQH2 into the IMS. One electron from a second UQH2 is used to reduce cyt-c, and the other is used to regenerate a UQH2 in the membrane from the semiquinone radical produced earlier, with uptake of protons from the matrix. This is again accompanied by the release of two protons per UQH2 into the IMS. In upshot, only one net UQH2 has been oxidised, but four protons have been pumped.
Cytochromes are small proteins containing a haem group (much like myoglobin or haemoglobin). They are grouped into three types (a, b and c) according to the type of haem and how it is bound into the protein.
Cytochrome-b proteins contain an iron protoporphyrin-IX prosthetic group, which is bound by dipole interactions. The Fe ion is hexacoordinated: 4 ligands from the N's of haem, and 2 from the histidines in the protein.
Cytochrome-c contains a haem-c prosthetic group bound covalently by its ring to cysteines in the protein. The Fe ion is hexacoordinated: 4 from the N's of haem, 1 from a histidine in the protein, and 1 from methionine in the protein.
Cytochrome oxidase dumps electrons from cytochrome-c onto oxygen, generating water, and pumping one proton per cytochrome-c.
Complex IV is more commonly termed cytochrome oxidase (or even just cyt-ox). It pumps 2 H+ per 2 cyt-cRED, and produces 1 H2O per 2 cyt-cRED oxidised. Complex IV receives its electrons from cytochrome-c, which is a small, mobile protein that diffuses from complex III to complex IV. The electrons are passed through a number of cytochrome-a and copper ion centres. CuB and cyt-a3 actually perform the reduction of oxygen to water. Each NADH originally oxidised yields 2 electrons, and these are enough to reduce half an O2 molecule to H2O (i.e. four electrons - two NADH - are required to reduce a whole molecule of dioxygen).
Cytochrome-a contains a haem-a prosthetic group bound by 'hydrophobic forces' to the protein. It also has a long phytol tail (just like chlorophyll). The Fe ion is pentacoordinated: 4 from the N's of haem, 1 from a histidine in the protein. This leaves a binding site for oxygen.
The reason that electrons flow through the various complexes is that earlier stages have lower redox potentials, so can provide electrons for downstream reactions.
The rotation of F0 can be seen by attaching a fluorescent actin tail to the F0 subunits.
Complex V is ATP synthase (an F-type ATPase). It converts an H+ gradient into ATP, producing c. 1 ATP per 3 or 4 H+ (stoichiometry still not quite certain). It actually acts like a motor: the FO subunit rotates as protons flow through and ATP is synthesised due to the conformational changes this causes in F1. It probably requires 3 protons to actually form one molecule of ATP, but one further proton is required to translocate ATP out of (and ADP/phosphate into) the matrix.
As protons flow through the a/b subunits (the stator) of FO, they force the ring of twelve c subunits (the rotor) in the membrane to rotate. This rotation is transmitted to the γ/ε subunits (the stalk) of F1, which change the conformation of the α/β subunits (the headpiece) of F1, makinf ADP and phosphate react to form ATP inside the β subunits. The headpiece is prevented from rotating by the binding of δ to the a/b stator, which is itself firmly anchored in the membrane.
The electron transport chain carries the electrons produced by the oxidation of NADH to NAD through complexes I, III and IV. This electron transport is used to drive proton pumping through the membrane. The electrons are eventually dumped onto oxygen, which is reduced to water. The proton gradient built up by these processes is used to drive the FOF1 ATPase (in reverse) to generate ATP. The oxidation of 1 NADH pumps (about) 10 protons. ATPase generates (about) 1 ATP from 4 protons.
2 ATP (substrate level phosphorylation)
2 ATP (substrate level phosphorylation)
2 NADH → 0 ATP
2 NADH → 5 ATP
2 ATP/GTP (substrate level phosphorylation)
8 NADH → 20 ATP
2 FADH2 (succinate) → 3 ATP
Approximate total yield
Take the energy budgets with a pinch of salt.
· NADH must be translocated into the mitochondrion from the cytoplasm: this costs ATP.
· The complexes are not 100% efficient.
· The inner membrane is not 100% impermeable.
· There is still argument about the stoichiometry of the various complexes.
Aerobic respiration is approximately 15 times more efficient than anaerobic. The P/O ratio (ATP made per oxygen atom reduced) is about 3 for NADH and 2 for succinate (FADH2). In books, you will find many different estimates of the ATP to glucose ratio, the number of protons pumped by each complex, the proton to ATP ratio for ATPase, etc. The numbers presented here are not to be taken as the definitive version!
Chemiosmosis is the name given to the generation of ATP from a proton gradient. It occurs in all living things:
The components of the chemiosmotic systems are similar too:
· Mitochondria, chloroplasts and purple-bacteria use a cytochrome complex to run the Q-cycle on quinones.
· The prosthetic group of cytochromes and chlorophyll are both porphyrins with phytanol tails.
Many of the proteins also contain
· Chloroplasts transfer electrons from water to NADPH, mitochondria transfer electrons from NADH to oxygen.
· All use an F-type ATPase in reverse to generate ATP.
Chemiosmosis can be disrupted by a variety of chemicals. In oxidative phosphorylation, some of these inhibitors are quite infamous:
Rotenone blocks electron flow from
· Antimycin-a blocks electron flow from cyt-bL to UQ in complex III (Q-cycle).
· Cyanide and carbon monoxide block access of O2 to cyt-a3 in complex IV.
· Oligomycin damages FO subunit of complex V.
These inhibitors were useful in the early research on the respiratory chain, and are still used to halt the chain at a particular point to study the stoichiometry of proton pumping.
Chemiosmosis works by generating a proton-motive force. The proton-motive force is the free energy associated with a gradient of protons across a proton-impermeable membrane. It is composed of two components: a chemical concentration gradient and an electrochemical charge gradient.
∆G = R T ln ( [H+]matrix ⁄ [H+]ims) − z F ∆Em
As well as simple inhibition, we can also uncouple electron transport from ATP synthesis by destroying the proton motive force. This is called uncoupling. Ionophores are rather good at this.
Dinitrophenol (DNP) is a proton ionophore (a weak acid). It carries protons across a membrane in a similar way to valinomycin with potassium ions. 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.
Other ionophores are more specific. Valinomycin is a potassium ionophore: it destroys ∆Em but not ∆pH: It uncouples ATP synthesis in mitochondria but not in chloroplasts, indicating that mitochondria use ∆Em (−150 mV), but not ∆pH (usually only about 0.5 pH units). Nigericin is an antiport ionophore that swaps H+ for K+. This is charge-neutral, so destroys ∆pH but has no effect on ∆Em. Nigericin effectively uncouples chloroplast ATP synthesis, but not mitochondrial, indicating that chloroplasts use ∆pH (usually about 4 units: stroma at pH 8, lumen at pH 4), but not ∆Em (0 mV).
Sometimes, organisms want to generate heat rather than ATP from chemiosmotic gradients. Brown fat tissue is mitochondria-rich adipose tissue. Lipid is oxidised and a proton gradient built up, but this is uncoupled from ATP synthesis by thermogenin. Thermogenin is a proton channel found in brown fat mitochondria. The flow of protons through the membrane generates heat.
Plants have a number of interesting 'extras' in their mitochondrial membranes. They have an intermembrane-space side NADPH dehydrogenase and a matrix-side rotenone-insensitive NADH dehydrogenase. They also have an alternative oxidase (alt-ox) that uncouples electron transport from ATP synthesis. This system (in theory) could completely uncouple NADH oxidation from ATP production, generating almost nothing but heat from the Krebs cycle.
What is the alternative oxidase for? It is known to be under hormonal control; it is stimulated by the plant hormone salicylic acid (Aspirin). It produces heat and could allows Krebs cycle (and the associated amino-acid pathways) to run even if ATP is not required by the cell (the energy overspill hypothesis). It also removes oxygen and prevents the build-up of reactive oxygen species produced by respiration and photosynthesis. However, in most cases, no-one really knows what it is 'for'. However, in aroids (arum lilies) it is know that the alternative oxidase generates heat in their inflorescence, volatilising amines and other fly-attracting chemicals. Skunk cabbage uses the heat generated to escape snow burial of its flowers.
You may find this diagram useful:
1. Why does the oxidation of succinate produce less ATP than that of malate?
2. Why do mitochondria contain glutathione?
3. Complete the table:
4. Which intermediates would you expect to accumulate if you treated mitochondria with antimycin-A?
5. Valinomycin uncouples electron transport from ATP synthesis, but it is not a proton ionophore. How does it work?