Mitochondria and Cellular Oxidation | Term Paper | Cell Biology

Read this term paper to learn about the role of mitochondria in cellular oxidation.

The carbon chains oxidised in cellular respiration originate from carbohydrates, fats, and proteins. As a preliminary to cellular oxidation, these three types of molecules are first converted into smaller units.

Polysaccharides are hydrolysed into individual monosaccharides, fats are hydrolysed into glycerol and fatty acids, and proteins are split into amino acids. These products monosaccharides, glycerol, fatty acids, and amino acids-are the immediate fuels for cellular oxidation.

These fuels are oxidised in a series of reactions that may include one or both of two major stages, the first occurring outside mitochondria and the second inside. In the first stage, called glycolysis, fuels are partially oxidised and converted to shorter three-carbon segments, which are completely oxidised to CO₂ and water in the second stage. Most of the ATP generated in cellular oxidation is produced in the second stage.

Monosaccharides and other carbohydrate units follow a main line of oxidation through both stages. Glycerol enters the glycolytic pathway in the first stage, and the fatty acids enter mitochondria for oxidation in the second stage. Amino acids are deaminated (the amino, or —NH₂ group is removed) and converted into molecules that may enter the pathway in either stage.

Cellular Oxidation:

In the following discussion, it is important to remember that oxidation does not require direct combination of molecular oxygen with a metabolite. Oxidation describes any reaction in which electrons are removed from a molecule. For every oxidation, an electron acceptor is reduced by combining with the removed electrons.

Therefore, each oxidation is accompanied by a reduction. Frequently oxidation involves removal of one or two hydrogen ions (protons) as well as electrons. The electron acceptors reduced during cellular oxidation may also combine with one or both of these hydrogens as well as with the electrons removed.

The amount of energy associated with the removed electrons depends on the orbitals they occupied in the oxidised molecule. Some of this energy is used to drive ATP synthesis in glycolysis and mitochondrial oxidations. The energy of the removed electrons can be expressed as a relative potential or voltage by comparison with an arbitrary standard. The standard used is the characteristic energy of electrons removed from hydrogen in the reaction H₂ → 2H⁺ + 2e^– catalysed by platinum.

The potential, or voltage, assigned to these electrons is 0.00 V. All other potentials, called redox or reduction-oxidation potentials are measured and assigned a value with respect to this standard. The relative potentials of the electrons removed from me important intermediates in cellular oxidation.

The First Stage of Cellular Oxidation: Glycolysis:

The overall reactions of glycolysis split glucose and other six- carbon monosaccharides into three-carbon fragments. During glycolysis a single oxidation occurs, and each molecule of glucose yields a net gain of two ATP. Glycolysis has two major parts. In the first part, six- carbon molecules derived from glucose are raised to higher energy levels at the expense of ATP.

This part of the sequence in effect raises the derivatives of glucose to energy levels high enough to enter the second part. In the second part, the energy expended in the first part is recovered with a net gain in ATP through oxidation of the high energy derivatives of glucose. In the process, the glucose derivatives are split into two three-carbon units of pyruvic acid.

Each step in the glycolytic pathway is catalyzed by a specific enzyme, and the entire series of reactions involves 1 I enzymatic protein. The various enzymes, reactants, and products of glycolysis are in solution in the cytoplasm outside mitochondria and apparently interact through random collisions.

The Reaction of Glycolytic Sequence:

The individual reactions in the glycolytic sequence and the enzymes are catalysing them. In the first three reactions of the pathway, glucose is converted into a more reactive derivative containing two phosphate groups. Two molecules of ATP break down to provide the energy required for the phosphory action of glucose.

The activity of the enzyme catalysing the first reaction of the pathway, liexokinase, illustrates one of the many controls regulating the rate of oxidation in cells. The product of the first reaction, glucose- 6-phosphate, is an inhibitor of hexokinase. If glucose- 6-phosphate accumulates because be remainder of the sequence is running slowly, the hexokinase enzyme is inhibited, blocking further entry of glucose into the pathway.

Similar regulatory mechanisms control glycolysis as later points in the sequence. The glucose-6-phosphate produced in the first step is rearranged and then phosphorylated into fructose-1, 6-diphosphate at the expense of a second ATP. The characteristics of this reaction illustrate another mechanism regulating glycolysis, this one linked to the ATP supply.

The enzyme catalysing the phosphorylation, phosphofructokinase, is inhibited by high concentrations of ATP and is stimulated by ADP and inorganic phosphate. If sufficient ATP is present in the cytoplasm, phosphofructokinase is inhibited and the subsequent reactions of glycolysis slow or stop.

A surplus of other products of oxidative metabolism, such as reduced NAD, also inhibits the enzyme. If energy- requiring activities like place elsewhere in the cell, resulting in the conversion of ATP to ADP and phosphate, the accumulation of ADP and inorganic phosphate stimulates phosphofructokinase, increasing the rate of glycolysis and ATP production.

Regulation by phosphofructokinase is probably the most sensitive and significant of the controls of glycolysis, since it is directly keyed to the relative concentrations of ADP and ATP and thus to the late at which cells use energy for their activities.

The remaining reactions of glycolysis proceed without ATP. Fructose- 1, 6-diphosphate is broken into two different three-carbon segments, which are readily interconverted, forming a “pool” of the two three- carbon sugars. One of these sugars, 3-phosphoglyceraldehyde (3-PGAL) enters into the remaining steps in glycolysis. As 3-PGAL is depleted from the pool, it is replaced by conversion of the second three carbon product.

At the next step in the sequence, two electrons and two hydrogens are removed from 3-PGAL. This oxidation is the primary energy-releasing step of glycolysis. Some of the energy released is trapped, as a part of the reaction, by the addition of a second phosphate from the medium (not from ATP) to form the product 1, 3-diphosphoglyceric acid. The electrons removed at this step have a relatively high potential or voltage and are accepted by a molecule called nicotinamide adenine dinucleotide.

NAD is a carrier molecule transporting high energy electrons between cellular reaction systems. The reduced NAD yielded at this step is one of the important high-energy products of the glycolytic sequence. The 1, 3-diphosphoglyceric acid product can also be regarded as a high energy molecule, since removal of its two phosphates later in the sequence releases large increments of free energy, much of which is captured in the conversion of ADP to ATP.

The reaction removing the first of the phosphates, because it illustrates one method by which the energy of oxidation is converted into a usable chemical form in the cell. If 1 mol of 1, 3-diphosphoglyceric acid is hydrolysed directly to the products 3 phosphoglyceric acid and inorganic phosphate, the reaction releases about 10,000 to 15,000 cal under standard conditions of temperature and pressure. In glycolysis, the breakdown of the diphosphorylated sugar is coupled to ATP synthesis.

The difference in calories between the two reactions represents energy captured in the formation of ATP. The 3-phosphoglyceric acid product then enters a series of reactions, including a rearrangement and a conversion in which the elements of a molecule of water are removed. In the final reaction of glycolysis, the remaining phosphate is transferred to ADP to produce ATP. The final reaction yields the end product of glycolysis, pyruvic acid, as well as ATP summarises the reactions of glycolysis.

Since each glucose molecule entering the pathway ultimately produces two molecules of 3-PGAL, a total of four molecules of ATP are produced in the conversion of 3-PGAL to pyruvic acid. The reactions attaching two phosphates to a glucose molecule in the initial steps require two molecules of ATP. Therefore, there is a net gain of two ATP, as well as two molecules of reduced NAD, for every glucose entering glycolysis.

The overall reactants and products of glycolysis are therefore:

Glucose + 2ADP + 2HPO₄^2- + 2NAD_ox → 2 pyruvic acid + 2NAD_red + 2ATP

In addition to a pair of high-energy electrons, each reduced NAD molecule carries one of the two hydrogen removed in the oxidation of 3-phosphoglyceraldehyde. The other hydrogen enters the pool of H⁺ ions in the medium surrounding the reaction sequence.

The two molecules of pyruvic acid yielded as final products are still relatively complex, energy-rich molecules that can be further oxidised to provide additional fret energy. These three-carbon molecules are the primary fuel for the second major series of cellular oxidations that take place in mitochondria.

Important Variations of the Basic Glycolytic Pathway:

The reactions of glycolysis from a central pathway in the metabolism of carbohydrates in both, plant and animal cells. Starch in plants and glycogen in animals are both long-chain polysaccharides made up from repeating glucose links to enter the glycolytic pathway after hydrolysis into individual glucose units.

Other sugars, including a wide variety of mono and-disaccharides, enter glycolysis after being converted enzymes into one of the initial molecules of the pathway. At the opposite end of the sequence, pyruvic acid may be modified to yield other products. In one of the most important of these modifications, pyruvic acid is converted into lactic acid after accepting electrons from the NAD reduced earlier in the sequence.

pyruvic acid + reduced NAD → lactic acid + oxidised NAD

The most significant feature of this modification is regeneration of oxidised NAD, which is then free to cycle back to accept electrons in the oxidation of 3-PGAL in glycolysis. Because oxidised NAD is continually regenerated by this alternate pathway, glycolysis can continue to run with the net production of ATP.

This pathway is vital to cells living temporarily or permanently without oxygen (reduced NAD normally transfers its electrons through a series of carriers to oxygen). This pathway occurs in the muscle cells of animals, including man, if intensive, sustained physical activity is carried out before increases in breathing and heart rate have a chance to meet the demand for oxygen in the muscle tissue.

The lactic acid accumulating as a by-product is oxidised later, when the oxygen content of the muscle cells returns to normal levels.

Another glycolytic variation occurs in organisms such as yeasts.

In this modification, pyruvic acid accepts electrons from reduced NAD and is converted by additional reactions into ethyl alcohol (two carbons) and CO₂:

pyruvic acid + reduced NAD → ethyl alcohol + CO₂+ oxidised NAD

This variation in the glycolytic pathway is of central importance in human economics. It forms the biological basis of brewing and baking, and provides the alcohol of importance to one industry, and the CO₂ of significance to the other.

These variations in the glycolytic pathway, in which the electrons carried by reduced NAD are traded off to an organic substance such as pyruvic acid, are collectively called fermentations. In the alternate path­way, the electrons carried from glycolysis by reduced NAD eventually reach an inorganic substance, molecular oxygen fermentations of various kinds, producing a wide variety of products, are used as an ATP source by many species of bacteria.

Some of these species, called strict anaerobes, are limited to glycolytic fermentations for ATP production and cannot use oxygen at any time as a final electron acceptor. Others can use either glycolytic fermentations alone or both glycolysis and reactions equivalent to mitocho­ndrial oxidations if oxygen is available. Bacteria in this category are called facultative anaerobes.

A number of species, termed strict aerobes, are unable to live by fermentation alone. Many cells of higher organisms, including the muscle cells of vertebrates, are facultative and can switch bet­ween fermentation and complete oxidation depending on their oxygen supply. Others are strict aerobes.

Many of the intermediate reactions of glycolysis are reversible and may go in either direction in response to high concentrations of either reactants or products. The reversible steps are indicated by those that are essentially irreversible are indicated by alternate enzymatic pathways are available in the cytoplasm for the irreversible steps, enabling glycolysis to run in reverse.

For example, the first reaction in glycolysis, the conversion of glucose to glucose-6-phosphate by hexokinase, is essentially irreversible. However, an alternate enzyme, glucose-6-phosphatase, can catalyse the reverse reaction. Reversal of the pathway is an important part of reactions synthesizing six-carbon sugars and starch in both plant and animal cells.

The Second Stage of Cellular Oxidations:

Oxidation and ATP Synthesis in Mitochondria:

In the second major stage of cellular oxidations, pyruvic acid, three-carbon molecule produced by the final steps of glycolysis, is completely oxidised to CO₂ and H₂O, releasing large quantities of free energy. The yield of ATP in these reactions far exceeds the amounts obtained from glycolysis. The reactions occur in three parts, all located in mitochondria. In the first, pyruvic acid is shortened into a two-carbon segment, and one molecule of CO₂ is released.

In the second the two carbon segment is completely oxidised to two molecules of CO₂. In the third part, the electrons removed in these oxidations travel through a series of electron carriers to reach oxygen. Much of the free energy released by this transfer is used to drive the synthesis of ATP. Because oxygen is the final electron acceptor for these reactions the oxidative activities of mitochondria are frequently termed respiration.

Part 1 of Mitochondrial Respiration:

Oxidation of Pyruvic Acid to Two-Carbon Segments:

After entering mitochondria, pyruvic acid is shortened to two- carbon segment in a cyclic series of reactions. These fictions remove two electrons, two hydrogens, and one carbon CO₂ from the pyruvic acid chain.

The two-carbon segment produced by the oxidation is an acetyl (-CH₃CO^–) group:

pyruvic acid – acetyl group + 2e^– + 2H + + CO₂

The two electrons and one of the hydrogens removed in this ion are transferred to NAD. The CO₂ and the remaining hydrogen are released to enter the surrounding medium. The two-carbon acetyl group is transferred to an acceptor called coenzyme A to produce the high-energy substance acetyl coenzyme A. Coenzyme A is another carrier molecule based on nucleotide structure, as are ATP and NAD.

Coenzyme A accepts and carries two-carbon acetyl units, as ATP carries phosphates and NAD transports electrons. Most of the free energy released in the oxidation of pyruvic acid is captured as chemical energy in the conversion of coenzyme A to acetyl coenzyme A.

The overall reaction cycle in pyruvic acid oxidation, thus yields as net products acetyl coenzyme A, reduced NAD, and CO₂:

pyruvic acid + oxidised NAD + coenzyme → acetyl co-enzyme A + reduced NAD + CO₂

All of the reactants and products in Reaction 7-6 are multiplied by a factor of 2 if pyruvic acid oxidation is considered as a continuation of glucose oxidation, since each molecule of glucose entering glycolysis produces two molecules of pyruvic acid.

Pyruvic acid oxidation is catalysed by a molecular aggregate containing five enzymes and a series of intermediate electron and hydrogen carriers. The reactions carried out by this multi-enzyme complex, worked out primarily in the laboratory of L.J. Reed, proceed. The enzyme carrying out the first steps of the cycle, pyruvic acid dehydrogenase, is another regulatory enzyme that tunes the rate of cellular oxidations to the cell’s energy needs.

High ATP concentrations inhibit the activity of pyruvic acid dehydrogenase, slowing or stopping pyruvic acids oxidation. The enzyme is also inhibited, directly or indirectly, by high concentrations of reduced NAD or acetyl coenzyme A. High ADP concentrations, in contrast, stimulate activity of the enzyme. The result is a sensitive control mechanism that closely matches the rate of pyruvic acid oxidation to the energy requirements of the cell.

The enzyme-acceptor complex carrying out pyruvic acid oxidation exists in mitochondria in the form of aggregates containing as many as 100 or more polypeptide chains, including many copies of the central enzyme of the complex, pyruvic acid dehydrogenase.

Bacteria able to carry out oxidation of pyruvic acid possess a similar complex. A recent analysis of pyruvic acid oxidation in E. coli indicates that as many as 60 polypeptide chains may be present in a single aggregate in this species, with a total molecular weight of 4,800,000.

Both organic products of pyruvic acid oxidation are high energy substances. The acetyl units carried by acetyl coenzyme A serve as the immediate fuel for the remaining oxidative reactions of mitochondria, and the electrons carried by reduced NAD represent potential energy that is eventually tapped off to drive the synthesis of ATP.

Part 2 Mitochondrial Respiration:

Oxidation of Acetyl Units to CO₂ in Mitochondria:

The acetyl group carried by coenzyme A is oxidised to CO₂ in a cyclic series of reactions first deduced in 1937 by a British investigator, Hans Krebs, who received the Nobel Prize for his brilliant work with cellular oxidation. In the cycle which is named for Krebs, there is a continuous input of two-carbon acetyl units as reactants and a continuous output of the products- CO₂, ATP, reduced NAD, and an additional electron carrier in reduced form, flavin adenine dinucleotide.

The molecules forming intermediate parts of the cycle are continuously regenerated as the cycle turns. Only one molecule of ATP is formed as a direct product of each turn of the Krebs cycle.

Most of the energy released by the several oxidations of the cycle is trapped in the electrons carried from the cycle by reduced NAD and FAD.

The reactions of the Krebs cycle proceed. In the first step in the sequence, the acetate group linked to acetyl coenzyme A is transferred to oxaloacetic acid (four carbons), forming citric acid (six carbons). This reaction releases coenzyme A, which is then free to recycle through another oxidation of pyruvic acid.

acetyl coenzyme A + oxaloacetic acid—citric acid -+ coenzyme A

The enzyme catalysing reaction, citrate synthetase, is one of the major regulators of the Krebs cycle. The enzyme is inhibited and the first reaction of the cycle slows or stops if succinyl -coenzyme A, a product of a reaction later in the cycle, accumulates. The enzyme is also inhibited by high concentration of ATP.

The citric acid product of the first reaction is rearranged in a series of steps into isocitric acid, which becomes the reactant for the first oxidation of the cycle. This oxidation is catalysed by either of two enzymes both of which are called isocitrate dehydrogenase. The reactions catalysed by are enzymes are identical except that one uses NAD and the other the closely related substance nicotinamide adenine dinucleotide phosphate as the electron acceptor.

Two electrons and two hydrogens are removed from isocitric acid in the oxidation; at the same time, the carbon chain is reduced in length from six to five carbons, yielding α-ketoglularic acid as product. The carbon removed is released as CO₂. Which of the two enzymes catalyse this reaction depends on the relative concentrations of ADP and ATP in the medium. The NAD-specific enzyme is more active than the NADP form when the ATP concentration is relatively low.

If ATP concentration is high, the NAD-specific enzyme is inhibited, and NADP is favoured. High concentrations of reduced NAD, which have a similar inhibitory effect on the NAD-specific enzyme, also favour NADP as the electron acceptor. On the other hand, high ADP concentrations stimulate the activity of the AD-specific enzyme, which actually requires ADP as a cofactor for its activity.

These differences are significant for ATP production because reduced NAD normally transfers its electrons to the electron transport system of mitochondria that generates ATP. Reduced NADP instead acts as an electron donor for reactions that build up more complex substances in which reductions are required. As a result of this system, isocitric acid oxidation is finely tuned to the needs of the cell for ATP.

Under the usual conditions, in which cellular activity demands a more or less continuous supply of ATP, the isocitric acid dehydrogenase enzyme using NAD as an electron acceptor predominates in Krebs cycle oxidation at this step, leading to ATP synthesis.

At the next step in the cycle, α-ketoglutaric acid is oxidised to succinie acid in a side cycle that closely resembles pyruvic acid oxidation. The enzyme-acceptor complex active in this reaction includes essentially the same types of enzymes and intermediate electron acceptors.

The central enzyme in the complex, however, is α-ketoglutaric acid dehydrogenase, which catalyses removal of two electrons and two hydrogens from α-ketoglutaric acid. At the same time, one carbon is removed from the acid and released as CO_2. The product, a succinyl group (four carbons) remains attached to the complex until its transfer to coenzyme A in a step analogous to reaction 3. This transfer forms the high-energy product succinyl coenzyme A.

The electrons and hydrogens are then transferred from acceptors of the complex to NAD, in a step equivalent to reaction 4. Coenzyme A is regenerated by a subsequent reaction in which the succinyl group is removed, releasing succinic acid and a large increment of free energy. Much of this free energy is captured in the synthesis of a molecule of ATP, the only ATP, originating directly from the Krebs cycle.

succinyl coenzyme A + ADP + HPO₄² → succinic acid + coenzyme A + ATP

ATP synthesis proceeds directly as shown in this reaction in bacteria. In animals, the enzyme catalysing the reaction phosphorylates guanosine diphosphate (GDP) instead. However, the guanosine triphosphate (GTP) produced is subsequently converted to ATP by a mitochondrial enzyme that readily interconverts the various nucleoside triphosphates

GTP + ADP ⇋ ATP + GDP

As net products, the overall reaction sequence oxidising α-ketoglutaric acid thus yields,

α-ketoglutaric acid + oxidised NAD +ADP + HPO₄² → succinic acid + reduced NAD + ATP + CO₂

Succinic acid is oxidised at the next step of the cycle by the enzyme succinic acid dehydrogenase. The enzyme is unique among the various proteins catalysing the Krebs cycle because it is tightly bound to the inner mitochondrial membranes. There it forms part of the integral proteins of the cristae. Also found that the enzyme in the membrane is its own electron acceptor FAD. FAD is another carrier molecule resembling ATP, NAD, NADP, and coenzyme A in structure.

The combination of this acceptor with the enzyme is known as a flavorotein. In the reaction catalysed by succinic acid dehydroenase, FAD is reduced by accepting two electrons and both hydrogens removed from succinic acid. The product of the reaction is the four-carbon molecule fumaric acid,

succinic acid + oxidised FAD → fumaric acid + reduced FAD

The final oxidation of the Krebs cycle occurs after a rearranging reaction in which fumaric acid is converted to malic acid by the addition of the elements of a molecule of water. Malic acid is then oxidised to oxaloacetic acid. NAD acts as in electron acceptor for the reaction and also binds one of the two hydrogens removed from malic acid. The molecule of oxaloacetic acid produced replaces the oxaloacetic acid used in the first reaction of the Krebs cycle, and the cycle is ready to turn again, summarises the reactions of the cycle in simplified form.

Products of the Krebs Cycle:

We can now determine the overall products of the Krebs cycle and summarise the cellular oxidation of glucose. As the Krebs cycle proceeds through one complete turn, one two-carbon acetyl unit is consumed and two molecules of CO₂ are released. At each of four reactions in the cycle two electrons are removed. We will assume for this summary that the first oxidation of the cycle uses NAD rather than NADP as the electron acceptor.

At three of the oxidations, then, NAD is the acceptor, producing three reduced NAD molecules; one step produces a molecule of reduced FAD. As a part of the oxidation of α-ketoglutaric acid, one molecule of ATP is generated. Oxaloacetic acid, used in the initial reaction of the Krebs cycle, is regenerated in the final reaction.

Thus, as overall reactants and products, the Krebs cycle includes:

acetyl unit + 3 oxidised NAD + oxidised FAD + ADP+HPO₄² → 2CO₂ + 3 reduced NAD+ reduced FAD + ATP

With this information we can sum up all of the products of complete oxidation of glucose to CO₂, from glycolysis through the Krebs cycle. For each molecule of glucose entering the series, the Krebs cycle will turn twice. Glycolysis and pyruvic acid oxidation together yield four reduced NAD, two ATP, and two CO₂ molecules, since two molecules of pyruvic acid are oxidised for each glucose molecule entering the sequence.

Adding to this the products of two turns of the Krebs cycle for each glucose entering oxidation, we have:

glucose + 4ADP + 4HPO₄^2- + 10 oxidised NAD + 2 oxidised FAD → 4ATP + 10 reduced NAD + 2 reduced FAD + 6CO₂

Note that at this point little ATP has been produced. However, the electrons carried by the 10 reduced NAD and 2 reduced FAD molecules occupy orbitals at high-energy levels and contain most of the energy obtained from oxidation of the glucose to six CO₂ molecules. The free energy released in the transfer of these electrons to oxygen is used as the major source of energy for cellular ATP synthesis in the third part of mitochondrial oxidations.

The Carrier Molecules:

With one exception, all of the known electron carriers consist of a protein molecule in combination with a tightly bound, non-protein prosthetic group that is the actual electron carrier. The prosthetic groups are alternately oxidised and reduced as electrons flow through the system.

The single exceptional carrier, coenzyme Q, is a lipid-like substance suspended in the membrane interior without direct linkage to a protein. Although coenzyme Q and most of the prosthetic groups have been isolated and chemically characterised, the protein portions of the carriers, except cytochrome c, are integral membrane proteins that can be removed only by techniques that seriously denature them.

As a result, the protein segments of the carriers are relatively poorly known. Four major kinds of carriers have been identified in the chain. The flavoproteins, including FAD and flavin mononucleotide (FMN) are electron carriers with prosthetic groups based on nucleotides.

In FAD and FMN, the part of the prosthetic groups alternately oxidised and reduced during electron transport is derived from riboflavin, a vitamin of the B group. Since this prosthetic group contains a nitrogenous base, a five-carbon sugar, and a phosphate group, it is classified as a nucleotide. The flavoproteins carry electrons in pairs and also bind two hydrogens of form FADH₂ or FMNH₂ during reduction.

The cytochromes, including cytochrome a, a₃ b, c, and d have a complex porphyrin ring containing a central iron atom as a prosthetic group. The various cytochromes differ in minor substitutions in side groups attached to the porphyrin ring. In each, alternation between the oxidised and reduced forms occurs by means of a single electron gained or lost by the central iron atom, which can exist as either Fe²⁺ or Fe³⁺. In contrast to the flavoproteins, the cytochromes carry no hydrogens.

Cytochrome c is unique among the electron carriers in, that it is a peripheral membrane protein, easily removed from mitochondrial membranes without being denatured. As a result, cytochrome c is the best-known of the protein-based electron carriers.

Iron-sulfur proteins comprise the third kind of mitochondrial electron carrier. As yet, these proteins are poorly characterised and are known only to contain centers containing iron atoms in close association with- SH (thiol) groups. Presumably, the iron and sulfur atoms are part of a prosthetic group attached to the protein; in reduction and oxidation, the iron atoms are believed to alternate between Fe²+ and Fe³⁺ states.

At least seven different iron-sulfur proteins have been detected in the mitochondrial electron transport system. As far as is known, these carriers transport single electrons and hydrogens, in a pattern similar to that of the cytochromes.

The final kind of electron carrier, coenzyme Q contains a quinone ring that is alternately oxidised and reduced during electron transport. Attached to the ring is a hydrophobic side chain, which in mitochondria consists of a series of ten repeating subunits. Coenzyme Q is a dual electron-hydrogen carrier and has two redox levels or states in which it may accept either one electron and hydrogen or electrons and hydrogens in pairs. This characteristic figures in one of the latest modifications of Peter Mitchell’s cherniosmotic hypothesis.

Reconstructing the Electron Carrier Sequence:

A variety of biochemical techniques has been used to assign the known carriers to their probable locations in the electron transport chain. Part of this work is based on the measured redox potentials of the purified carriers or prosthetic groups. Electrons released from the various carriers have a characteristic energy; by arranging the carriers in order of increasingly positive potential, their sequence in mitochondrial electron transport can be approximated.

This method suffers from the fact that redox potentials measured under standard conditions, when the carriers are in isolated form, may differ from the potentials in the natural situation in the membrane, leading to the possibility of errors in placing them in sequence. A somewhat more direct method for sequencing the carriers is based on the fact that most of the carrier molecules undergo pronounced colour changes on conversion from oxidised to reduced form.

The flavoproteins, for example, are yellowish in solution when oxidised and colourless when reduced. These changes can be traced quantitatively by measuring the absorption spectra of the molecules in solution (or in mitochondrial membranes). In this technique, the amount of light absorbed by the carrier at each wavelength is measured and plotted. As colour changes occur on oxidation or reduction, characteristic absorption peaks appear and disappear in the spectrum plotted for the molecule.

These absorption spectra were used in the laboratory of critton Chance to place the carriers in a tentative sequence. Chance and his co-workers found that in intact, active mitochondria, most of the FAD or FMN molecules are in the reduced state. Two of the cytochromes, a and a₃, tend to be present almost entirely, in the oxidised state. Other carriers fell between these two extremes, falling into a natural sequence in terms of the ratio of oxidised to reduced forms.

Reasoning that the carriers appearing in highest proportion in the oxidised form were nearest oxygen in the sequence, Chance assigned each carrier a position in the electron transport system. This method is also not without pitfalls, since in intact mitochondria some of the absorption peaks of different carriers overlap, making interpretation of the spectra obtained difficult. Also, some of the carriers, such as coenzyme Q and the iron-sulfur proteins, cannot be readily detected or assigned definite positions by this technique.

These results were strengthened by Chance and others by combining the light absorption studies with the use of inhibitors known to combine with specific carriers and interfere with their oxidation or reduction. Carriers falling after the inhibited carrier in the sequence gradually become oxidised by removal of electrons and show the characteristic colours for the oxidised forms.

Carriers falling before the blocked carrier remain in the reduced form and show colours characteristic for this state. For example, the drug antimycin a blocks electron transport from cytochrome b. In mitochondria exposed to the drug, NAD, the flavoproteins, and cytochrome b remain in the reduced state, and cytochromes a and c become oxidised. This indicates that NAD and the flavoproteins fall before cytochrome b, while cytochromes a and c fall after it.

By using different inhibitors and nothing the “crossover points” between carriers converted to reduced or oxidised states, the tentative positions assigned by means of redox potentials and absorption spectra were strengthened. Combining the results of these approaches established the probable carrier sequence. Note that, electrons have two routes of entry into the chain, one at FMN and one at FAD, which join at coenzyme Q.

Electrons flow spontaneously along the chain, losing energy as they move from orbitals in one carrier to orbitals in the next. At some of the steps, sufficient energy is released to drive, the synthesis of ATP. Some of the carriers carry electrons in pairs, and some singly. Hydrogen combines directly only with NAD, FAD, FMN, and coenzyme Q and at the end of the sequence with oxygen.

When electrons pass from hydrogen to a non-hydrogen carrier, hydrogen is considered to be released to the medium as H⁺ ions or, in the case of the Mitchell hypothesis, to be expelled across the inner mitochondrial membranes. At the level of the final acceptor, hydrogen ions reenter the sequence in the reduction of oxygen to water.

Because of the ambiguities in the different sequencing techniques, there are still uncertainties about the placement of some of the carriers. These difficulties are most pronounced in the assignment of the iron- sulfur proteins. The total number of these carriers and their positions in the electron transport sequence are still controversial. Because of overlapping spectra in the light wavelengths absorbed by cytochrome b, it is unclear at present whether there are one, two, or three different molecules of this cytochrome transferring electrons between coenzyme Q and cytochrome c₁.

Similarly, it is presently unknown whether the two prosthetic groups identified as cytochromes a and a₃ are carried on the same protein molecule or different proteins. Because a and a₃ are always closely associated, the consensus at the present time is that they combine into one molecule, presumably in the form of two porphyrin rings, one of the a and one of the a₃ type, carried in crevices in a single protein.

Electron Transport and ATP Synthesis:

During the late 1940s, A. L. Lehninger and his associates established that transport of electrons along the mitochondrial carrier system, from reduced NAD to oxygen, is directly linked to ATP synthesis. Lehninger showed that isolated mitochondria treated to make them permeable to NAD could synthesize ATP if placed in a medium containing reduced NAD, ADP, phosphate, and Mg²⁺ ions. For each molecule of reduced NAD added, the isolated mitochondria synthesized three molecules of ATP.

When ascorbic acid, an electron donor incapable of reducing carriers higher in the sequence than cytochrome c, was added to the isolated mitochondria, only one phosphorylation of ADP to ATP occurred for each molecule of ascorbic acid. This showed that energy sufficient to drive the synthesis of one molecule of ATP is released somewhere in the sequence from cytochrome c to oxygen.

Other possible sites of significant energy release were located by an extension of the light absorption studies used to sequence the carriers. Chance and G.R. Williams used a mitochondrial preparation having a limited supply of ADP and an excess of oxidisable substrate.

The limited supply of ADP caused bottlenecks at three sites in the electron transport system, which was detectable by colour changes indicating an excess of reduced NAD, cytochrome b, and cytochrome c. This indicated, for example, that the step in electron transfer from NAD to flavoprotein is closely coupled to the synthesis of one ATP molecule; a limited supply of ADP causes the accumulation of reduced NAD.

More recently, the findings from these studies have been supplem­ented by the results of studying isolated parts of the electron transport chain after disruption of mitochondrial membranes by sonication or detergents.

By this approach, carried out by D.E. Green, E. Racker and his associates, and others the electron transport chain was broken into segments containing several carriers or into individual carrier molecules. Reassembling the carriers in various combinations with membrane segments and the ATP-Synthesizing enzyme then allowed an evaluation of the capacity of parts of the electron transport system to drive ATP synthesis.

The combined results of these techniques indicate that ATP is synthesized in the mitochondrial transport chain as pairs of electrons pass from NAD to coenzyme Q (site I), from cytochrome b to cytochrome c (site II), and from cytochrome a-a₂ to oxygen. Thus, each pair traversing the transport system from reduced NAD to oxygen generates three ATP molecules. Since the electrons carried by FAD enter the chain at a point past site I, the first ATP generating site, the electrons carried by reduced FAD from succinic acid oxidation give rise to only two ATP molecules.

Synthesis of ATP in the isolated and reassembled transport systems occurs only if three basic conditions are met:

(1) The various carriers must be combined with phospholipids into bilayers or intact inner mitochondrial membranes;

(2) The membranes must be closed into sealed vesicles with an inside space completely separated by intact membranes from the medium outside the vesicles; and

(3) The mitochondrial ATP synthesizing enzyme must also be present in the sealed membranes of the vesicles with the carrier molecules. All of these conditions are satisfied in intact mitochondria.

A Summary of Glucose Metabolism:

With this information, the total number of ATP molecules synthesized for each molecule of glucose oxidised to CO₂ and H₂O can be calculated. Complete oxidation of one molecule of glucose (through glycolysis, acetyl coenzyme A formation, and the citric acid cycle) results in a total of 4ATP, 10 reduced NAD, 2 reduced FAD, and 6CO₂ molecules.

The ten electron pairs carried by reduced NAD, traversing the electron transport system, will result in the synthesis of 10 × 3 = 30 molecules of ATP. Reduced ADF, carrying electrons that enter the transport system at a later point, will cause the synthesis of 2 × 2 = 4 molecules of ATP.

Added to the total the overall oxidation of glucose yields 38 molecules of ATP:

glucose + 38ADP + 38HPO₄² + 6O₂ → 6CO₂ + 44H₂O + 38ATP

This total of 38 ATP assumes that the two molecules of NAD reduced in glycolysis will each induce synthesis of three ATP molecules inside the mitochondrion. However, reduced NAD from glycolysis cannot directly enter the mitochondrion to pass electrons to the transport system because the mitochondrial membranes are impermeable to NAD. Instead, the electrons carried by reduced NAD outside mitochondria are transferred to other substances that act as electron shuttles between the mitochondrial exterior and interior.

Two of these shuttle mechanisms are known. One is more efficient and results in the transfer of electrons from NAD outside to NAD inside the mitochondrion. The second is less efficient and results in the reduction of FAD inside the mitochondrion. If the more efficient shuttle is predominant, significant energy loss occurs, and 38 ATP will result from each molecule of glucose completely oxidised.

If the efficient shuttle predominates, the electrons from reduced TAD are transferred instead to FAD inside the mitochondrion. These enter the electron transport system farther long in the chain and result in the synthesis of 2 ATP for each molecule of reduced FAD. In this case, complete oxidation of glucose will result in a total of 36 ATP molecules instead of 38 which shuttle predominates depends on the articular species and tissues involved.

Under standard conditions (pH 7, reactants and products at 25°C and I M), the hydrolysis of ATP to ADP yields 7000 -al mol. Using this value as the energy required to synthesize ATP from ADP and HPO₄^2- , the total energy trapped during the oxidation of glucose may amount to either 36 × 7000 = 252,000 cal/mol or 38 × 7000 = 266,000 cal/mol. Combustion of glucose in air yields 686,000 cal/mol. On this basis, the efficiency of glucose metabolism in cells falls between 37% and 39% (252/686 × 100 and 266/686 × 100).

The energy captured under the temperature and concentration conditions in the living cell may actually be considerably higher than these figures indicate. The free energy change for the hydrolysis of ATP increases in magnitude at the concentration of the reactants and products is reduced. The concentration of ADP and ATP at the site of phosphorylation is not known with certainty.

Estimates of the actual free energy change vary from 9000 to 15,000 cal/mol depending on the, concentrations assumed to, occur in the cell. Using these figures gives a higher efficiency for the total oxidation of glucose, from about 50% at 9000 cal/mol to 80% at 15,000 cal/mol.

Where the actual figure falls in this range is unknown, but the efficiency is probably higher than the 37-39% calculated from standard conditions. Even at the 37-39% level, the efficiency of mitochondrial energy conversion is considerably higher than that of most energy-conversion systems designed by human engineers, which rarely perform above the 5-10% efficiency level.

ATP Synthesis in Mitochondria:

The research effort to unravel the mechanism coupling ATP synthesis to mitochondrial electron transport called oxidative phospho­rylation is presently among the most extensive and active in cell biology.

Experimentation, hypothesis, and speculation in this field have concentrated on two areas:

a. Characterisation of the ATP-synthesizing enzyme and its mechanism of action, and

b. Determining how the energy released in electron transport is used to power ATP synthesis.

The experiments and ideas connected with this field of research (by scientists who are sometimes called “mitochondriacs”) are among the most elaborate in contemporary biology. Understanding them requires extra effort; if you become confused, you are in good company. Eftaim Racker, one of the foremost workers in the field, once said in a talk that “anyone who is not confused about oxidative phosphorylation just does not understand the situation.”

The ATP-Synthesizing Enzyme of Mitochondrial Electron Transport:

Extraction and purification of mitochondrial membrane fractions reveals that the enzyme catalysing the synthesis of ATP in oxidative phosphorylation is embedded in the cristae membranes, in the same general location as the electron transport carrier molecules. Work by A.E. Senior, E. Racker, and others have shown that the enzyme is contained in a complex consisting of ten or more polypeptides.

Some of these polypeptides function to anchor the complex in the membrane, and some directly catalyse the reaction sythesizing ATP:

ADP + HPO₄^2- ⇋ ATP + H₂O

Racker’s work has allowed morphological identification of the ATPase enzyme with a lollipop-shaped particle that under some conditions can be seen extending from the inner mitochondrial membrane into the mitochondrial matrix. In Racker’s experiments with the particles, carried out with L.L. Horstman, mitochondria were isolated and mechanically disrupted by sonication or shaking in a medium containing glass beads. This treatment ruptures both the outer and inner mitochondrial (cristae) membranes. The raptured inner membranes seal spontaneously into closed vesicles capable of electron transport and ATP synthesis.

The inner membrane preparation, if viewed in the electron microscope after negative staining, shows the typical lollipop particles. Racker and Horstman treated isolated membranes of this type by exposing them to urea, a chemical that disrupts hydrogen bonds. After this treatment, the membranes were still capable of electron transport but could not synthesize ATP.

Examination after negative staining showed that the lollipop particles were missing from the membrane surfaces. The removed particles, if separately purified, were found to be capable of ATP hydrolysis, that is, of running reaction in reverse. Thus, the removed particles contained the ATPase activity associated with the inner membranes in intact mitochondria. Adding the purified particles back to the membrane preparations restored both the lollipops and the capacity for electron- transport-driven ATP synthesis to the membrane preparation.

Analysis of the inner membrane particles reveals that the ATPase activity occurs in the spherical “headpiece” of the lollipop. At least five different polypeptides can be identified with the headpiece, which is connected to the membrane by a “stalk” containing at least two poly­peptides. Anchoring the headpiece and stalk to the membrane is the “base,” containing an additional three or four hydrophobic polypeptides normally embedded deeply in the membrane.

The headpiece is easily removed from the stalk and base by various mild treatments, such as exposure to urea or low salt concentrations, indicating that in intact membranes, it is a peripheral protein. The lollipop structures are made visible most readily by negative staining. The particles are rarely seen in thin-sectioned mitochondria or in freeze- fracture preparations. As a result, it is not certain whether the particles actually extend from the inner membrane surfaces in intact mitochondria or are somehow extruded from a normal location closer to the membrane surface by the isolation or staining technique.

The Mechanism Coupling ATP Synthesis to Electron Transport:

In intact mitochondria, ATP synthesis is tightly coupled to electron transport. If electrons move through the system, ATP is synthesized; if no ADP is available, thus blocking ATP synthesis, electrons cannot pass along the carriers from NAD or FAD to oxygen. The nature of the coupling mechanism has been debated for many years and is still the subject of much controversy. Two major hypotheses are now considered seriously.

One is the chemiosmotic hypothesis of Peter Mitchell, which has steadily gained experimental support from its first statement in 1961 until the present; its basic tenets have been accepted by most investigators in the field. The second model, the conformational hypothesis, first advanced by P.D. Boyer in 1963, has gained recent attention because it can account for certain details not adequately explained by the chemiosmotic hypothesis.

The Chemiosmotic Hypothesis:

Mitchell’s hypothesis proposes that mitochondrial ATP synthesis is driven by a gradient of H⁺ ions set up directly by electron transport. According to Mitchell, H⁺ ions are expelled to one side of the membrane as electrons travel from NAD or FAD to oxygen. The H⁺ gradient established by the electron transport acts as a source of free energy that drives the synthesis of ATP from ADP and HPO₄^2- as the gradient runs down. Establishing the H⁺ Gradient:

The H⁺ gradient, according to the most recent modifications of the chemiosmotic hypothesis, depends on the fact that some of the carriers in the electron transport chain carry both hydrogens and electrons during their cycles of oxidation and reduction, while some carry electrons only.

In the first steps in the mechanism, reduced NAD (NADH) passes its two electrons and one hydrogen to FMN. Since FMN carries hydrogens in addition to an electron pair, a second H⁺ is absorbed from the medium inside the matrix to form FMNH₂. This carrier passes its electrons to the next carrier group in the chain, the iron- sulfur proteins. Because the iron-sulfur proteins are “pure” electron carriers, the hydrogens carried by FMNH₂ are released. Release occurs into the inter-membrane compartment, contributing the first two H⁺ ions to the gradient.

The next series of steps involves coenzyme Q in a mechanism that Mitchell calls the Q cycle. In the first step of the Q cycle, the two electrons carried by the iron-sulfur proteins are passed to two molecules of coenzyme Q, one to each Q molecule. These take up one H+ each from the matrix at the same time and go to the QH, or semi Quinone form. To go to the fully reduced, or QH₂, form the two Q molecules each accept an electron from a cytochrome b molecule (the source of the electrons carried by the two cytochrome b molecules) required for this transfer will soon be apparent.

The additional pair of H⁺ ions required is taken up from the matrix. Thus, in going from Q to QH₂, the two coenzyme Q molecules absorb 4H from the matrix. In the next step in the Q cycle, the two coenzyme Q molecules pass one electron each to the next carriers in the chain, two molecules of cytochrome c. Since the cytochromes are pure electron carries, the coenzyme Q molecules, in going from QH₂ to QH, release one H⁺ each to the inter-membrane compartment.

The second electron carried by each QH molecule, according to Mitchell, passes to each of two cytochrome b molecules. As this transfer takes place, the two coenzyme Q molecules go to the fully oxidised or Q form and release their last H⁺ ions, one each, to the inter-membrane compartment. A total of 6H⁺ is thus transferred from the matrix to the inter-membrane compartment as a pair of electrons passes from reduced NAD to this point in the chain.

The electrons carried by cytochrome c then move along the chain through a-a_$ cytochromes to oxygen. As the electron pair is accepted by an oxygen molecule, an additional 2H is removed from the matrix, converting 1/2C₂ to H₂O. The electrons passed to cytochrome b are ready to enter another Q cycle.

In cycling from the Q to the QH₂ form each coenzyme Q molecule thus picks up one electron from an iron-sulfur protein and one from cytochrome b. In cycling back from QH₂ to Q, each coenzyme Q passes one electron to cytochrome c and returns one to cytochrome b. The Q cycle and the movements of coenzyme Q shuttling H⁺ ions across the membrane from the matrix to the outside are considered to rest on the relatively small size of coenzyme Q and its solubility in the hydrophobic membrane interior.

Synthesis of ATP:

The hypothesis goes on to explain how the H⁺ gradient established by electron transport powers ATP synthesis. The ATPase complex is considered to consist of two parts, F₁, located on the membrane surface facing the matrix, and F₀, located within the membrane, F₁ contains the site catalysing ATP synthesis or hydrolysis, F₀ is proposed to contain a channel that allows H⁺ ions to approach the active site of the ATPase enzyme from the opposite side of the membrane. The membrane is otherwise considered to be impermeable to H⁺ ions.

The H⁺ gradient established by electron transport results in net movement of H⁺ ions from the inter-membrane compartment into the F₀, channel, through which they approach the active site of the enzyme. On the matrix side of the membrane, the active site of the enzyme binds ADPOH and POH from the medium, converting them to the ionised forms ADPO^– and PO^–. The 2H⁺ produced by this conversion are released into the matrix. The protons in the membrane F₀ channel now interact at the active site of the enzyme, removing one oxygen from PO to form water, which diffuses back to the opposite side of the membrane. Removal of the oxygen causes conversion of ADPO^– and PO^– to ADPOP, that is, to ATP.

ADPO^– + PO^– + 2H → ADPOP + H₂O

Since 2H⁺ are released into the matrix by the conversion of ADPOH and POH to ADPO^– and PO^– and 2H⁺ are removed from the opposite side by combination with oxygen to form H₂O, the overall effect is the equivalent of moving H⁺ from the side of high H⁺ concentration in the inter-membrane compartment to the side of low H⁺ concentration in the matrix, thus relieving the gradient. As a result, the total reaction tends to run in the direction of ATP synthesis as long as H⁺ is available in excess in the inter-membrane compartment.

Experimental Support for the Mitchell Hypothesis:

Mitchell’s chemiosmotic hypothesis, at first largely rejected by the scientific community, has met with such complete experimental support that there now seems little doubt that its two basic assumptions are correct:

(1) Electron transport creates a gradient of H⁺ ions, and

(2) The H⁺ gradient drives ATP synthesis.

The hypothesis and the new insights into bio-energetic that is provides have revolutionised scientific thinking in oxidative phosphorylation, photosynthesis, and active transport. Only in some details does Mitchell’s hypothesis, for which he received the Nobel Prize in 1978, remain open to question.

The electron transport creates an H⁺ gradient is clearly supported by the available evidence. The Racker laboratory showed that closed vesicles can be isolated from mitochondria in either right-side-out or inside-out form. The sidedness of the vesicles can be easily identified by noting whether the ATPase lollipops are carried on the inside or outside surfaces of the vesical membranes.

If the vesicles are right side out, with the lollipops on the inside surface (analogous to their position in mitochondria, in which they extend into the matrix), they expel H⁺ ions to the outside during electron transport. If inside out, with the ATPase lollipops on the outside surface, the vesicles concentrate H⁺ ions on the inside during electron transport. Thus H⁺ ions are actually expelled across mitochondrial membranes to the side opposite the ATPase enzyme as electron transport takes place, as proposed in the model.

Creation and maintenance of the H⁺ gradient requires that the inner mitochondrial membranes be impermeable to H⁺. Experimental support for this part of the hypothesis has come from Mitchell’s laboratory. Mitchell and J. Moyle set up a pH gradient across mitochondrial membranes and rioted the rate at which H⁺ could diffuse across the membranes to neutralise the gradient in the absence of electron transport and ATP synthesis. The rate of movement noted was so low that the inner mitochondrial membranes can indeed be regarded as essentially impermeable to H⁺ ions.

Support for the second basic proposition of the hypothesis, that the H⁺ gradient established by electron transport, drives ATP synthesis, comes from a number of sources. The first is the simple observation that closed, intact vesicles are necessary for ATP synthesis to occur in inner membrane preparations isolated from mitochondria. If the membranes are broken or made leaky, thus destroying any possibility of an H⁺ gradient, ATP synthesis stops.

The second comes from observations of the effect of uncouplers of additive phosphorylation. These substances, when added to mitochondria or mitochondrial vesicles, separate electron transport from ATP synthesis. Under the action of the coupler, electron transport runs continuously with no relationship to ATP synthesis.

With few exceptions, the couplers, such as DNP (2, 4-dlmitrophenol), are lipid-soluble agents that cause membranes to be leaky to H⁺ ions., H⁺ gradient removes the driving force for FP synthesis and stops phosphorylation, in accordance with the Mitchell hypothesis.

More direct evidence comes from experiments in which an imposed pH gradient was shown to cause ATP synthesis. The best of these was carried out in the laboratory of A. T. Jagenlorf, who used chloroplasts instead of mitochondria for several reasons. Chloroplasts contain an electron transport chain similar to the mitochondrial system, and ATP synthesis in chloroplasts is coupled to electron transport in exactly the same way as in the mitochondria.

The primary difference in ATP synthesis in the two organelles is in the source of high-energy electrons passing through the electron transport system. In mitochondria, these electrons come from oxidised fuel substances; in chloroplasts, their energy is derived from absorbed light. By placing isolated chloroplasts in darkness, Jagendorf could eliminate electron transport as a source of energy for ATP synthesis.

He then created a surplus of H⁺ ions inside the chloroplasts by placing them in a medium containing an acid that could penetrate inside. The chloroplasts were then transferred to a second medium at lower H⁺ concentration.

As the H⁺ ions inside moved outside in response to the gradient, ATP was synthesized. Since electron transport was eliminated as an energy source, the ATP synthesis could be ascribed directly to the artificially created pH gradient. Similar experiments were subsequently carried out by Mitchell and his co-workers with mitochondria.

Another light-driven system has also provided an elegant demonstration that an H⁺ gradient can provide the energy required for ATP synthesis. W. Stoeckenius discovered that the plasma membrane of a bacterium, Halobacterium halobrium, contains a pigment that resembles the visual pigments found in the retina of animal eyes.

The pigment, called bacteriorhodopsin, can change rapidly into either of two forms, one that absorbs light at a wavelength of 570 nm (“purple”) and one at 412 nm (“bleached”). At this pigment changes from the purple to the bleached form, it loses an H⁺ ion, and in the opposite change, it picks up an H⁺ ion.

Racker and, Stoeckenius found that purified rhodopsin, is combined with lipid bilayers in the form of closed vesicles, could take up H⁺ ions from the medium and concentrate them inside the vesicles when exposed to light. If the components of the ATPase complex were then added hydrophobic membrane proteins plus stalk and F₁ proteins, the vesicles were able to drive the synthesis of ATP when illuminated.

This simple system, containing nothing more than a closed membrane, a molecule establishing an H⁺ gradient, and the ATPase enzyme, provides conclusive evidence that H⁺ gradients can provide the energy required for ATP synthesis.

Experiments with ATPase-driven active transport systems also clearly support the idea that ion gradients can serve as energy sources for ATP synthesis. Normally the ATPase driven pumps, such as the Na⁺, K⁺-ATPase system of the plasma membrane, break down ATP and use the energy released to establish an ion gradient.

However, the pumps can be made to run in reverse- Imposing an artificially high ion gradient can cause the pump’s enzymatic system to run backwards and synthesize ATP from ADP and inorganic phosphate. These experiments directly show that the energy of an ion gradient can be used to drive ATP synthesis.

Taken together, the various experiments testing the chemiosmotic hypothesis establish beyond any reasonable doubt in mitochondria electron transport sets up an H⁺ gradient, and that the gradient in turn provides the energy source for mitochondrial ATP synthesis.

Problems with Details of the Mitchell Hypothesis:

While the major and basic tenets of the chemiosmotic hypothesis are clearly supported by experiment, certain details of the model remain controversial. Most prominent among these is the Q cycle mechanism for expelling H⁺ ions across the mitochondrial membranes. As of yet no evidence supports the unusual arrangement and sequence of electron carriers necessary for this mechanism.

Other difficulties stem from Mitchell’s assumption that 6H⁺ will be expelled across the membrane for each electron pair traversing the entire chain from NAD to oxygen. Actual measurement indicates that 6H⁺ may be expelled for each electron running through the carrier sequence, or 12H⁺ per electron pair.

In another sense, however, finding a higher ratio than predicted supports other, more basic tenets of Mitchell’s model. One persistent trouble with the model has been that, with only 6H⁺ expelled for each electron pair traversing the chain, most calculations (except for Mitchell’s) have shown that the H⁺ gradient created would not be quite high enough to power synthesis of the three ATP molecules formed when a pair of electrons travels the entire route from NAD to oxygen.

Increasing the level to 4-5 H⁺ expelled per electron, or 8-10 H⁺ per electron pair, would build a gradient high enough to satisfy all calculations of the energy required. These difficulties are relatively minor ones, however, and do not detract from the fact that in his brilliant hypothesis, Mitchell has successfully deduced the biochemical mechanism underlying oxidative phosphorylation.

The Conformational Hypothesis:

The conformational hypothesis of P.D. Boyer is of special interest because it provides a possible explanation for the details of ATP synthesis in the chemiosmotic hypothesis. Boyer’s idea is based on the model currently accepted for force generation in muscle. In muscle, myosin, a molecule with ATPase activity changes its conformation or folding pattern as it binds and hydrolyses ATP.

The conformational change imposes a strain on parts of the myosin, which is relieved by physical movement of a part of the molecule. The movement is multiplied by the millions of myosin molecules involved and is translated into movement of the entire muscle. In the muscle system, therefore, ATP breakdown is considered to cause strain and movement.

In Boyer’s application of this mechanism to oxidative phosphorylation, this process operates in reverse- Electron transport and the resultant H⁺ gradient lead to a conformational change in the mitochondrial ATPase, producing a strain in the structure of the molecule. This strain is relieved by the synthesis of ATP.

Conformational changes may be important in ATP synthesis as Boyer suggests. They may also be important in the mechanisms setting up the pH gradient through electron transport. For example, the expulsion of H⁺ across cristae membranes could be due to conformational changes in the carrier proteins.

In this case, a cycle of reduction and oxidation of a carrier molecule (or complex of carriers) could lead to a conformational change exposing an H⁺-bearing chemical group such as a carboxyl (—COON) at one surface of the membrane. In the new position, the group might dissociate more easily, leading to release of H⁺ at this side of the membrane.

Location of the Components of the Electron Transport and ATP-Synthesizing Systems:

The molecules carrying out electron transport and ATP synthesis were identified with the cristae membranes by experiments following the distribution of biochemical activity after disruption of mitochondria. At low concentration, a detergent such as digitonin breaks the outer mitochondrial membrane without damaging the inner membrane. The membranes can then be separately purified by centrifugation.

After removal of the outer membranes, the capacity for electron transport remains with the cristae membranes as long as intact, closed vesicles are retained. All of the various components of electron transport and ATP synthesis are found to be linked to the cristae membranes, rather than suspended in solution in the matrix.

Application of an extensive battery of newer techniques has begun to reveal the locations and arrangement of these components within the cristae membranes. The most productive of these methods utilise vesicles isolated from inner mitochondrial membranes oriented in right-side-out or inside out fashion.

These vesicles have been tested by three different probes:

(1) Antibodies developed against various components of the electron transport and ATP-synthesizing systems;

(2) Reagents that chemically modify or attach radioactive labels to the parts of proteins exposed on the inner or outer membrane surfaces, and

(3) Non-penetrating electron acceptors or donors that react specifically with individual carriers of the electron transport system.

None of the probes used can penetrate across the mitochondria] membranes. Basically, the probes test which of the two surfaces of the inner mitochondrial membranes, either the side facing the matrix (the M face) or the side facing the membrane space (the IM face), carry exposed portions of the molecules “recognised” by the probes.

The work with cytochrome a serves as a good example of the approach and the conclusions was drawn. Antibodies made against cytochrome a interact with the IM face, but not the M face, of vesicles derived from mitochondrial cristae.

Polylysine, a reagent capable of linking to the parts of proteins exposed at membrane surfaces, binds to cytochrome a only is applied to vesicles with the IM face exposed. From this information, cytochrome a is assigned an asymmetric location in the cristae membranes, in the membrane bilayer half facing the inter-membrane space.

Combination of the results from several laboratories, notably those of Racker and R.A. Capaldi reveals that most of the molecules functioning in electron transport and ATP synthesis share cytochrome as one-sided orientation and face only one of the two inner mitochondrial membrane surfaces. The single exception is succinic acid dehydrogenase, the enzyme carrying FAD as prosthetic group, which apparently spans the membrane and has parts exposed on both surfaces.

The active site of the enzyme, however, reacts with succinic acid only on the M face and is evidently directed towards this side. Locating coenzyme Q, the only electron carrier not linked to a protein, has proved difficult. Most experiments indicate that Q is buried entirely within the membrane. Chance, for example, observed that the quinone ring of coenzyme Q could react only with reagents possessing hydrophobic groups that can penetrate into the membrane interior.

Comparing the absolute ratios obtained when components of the electron transport system are extracted and purified reveals that the individual carriers occur in cristae membranes in the ratios 1 FAD: 1 FMN: 7-10 coenzyme Q: 2 cytochrome b: I cytochrome c₁: 2 cytochrome c: 2 cytochrome x: 2 cytochromc a₃.

Information from this and other approaches also indicates that there is one ATPase complex for each electron transport system. These ratios suggest that the carriers are coupled into units that contain the individual carriers in fixed proportions, together with one ATP synthesizing complex per unit.

If this is the case, it is interesting to note that the proportions of the carriers are apparently unrelated to whether the individual carriers transport electrons singly or in pairs. That is, single and pair electron carriers are not necessarily present in a 2: 1 ratio, as might be expected.

Location of Other Components of Oxidative Metabolism in Mitochondria:

All but one of the enzymes and substrates of the citric acid cycle, and the enzyme complex carrying out pyruvic acid oxidation, are released from the interior of mitochondria immediately on rupture of the inner mitochondrial membranes. Removal of the outer membrane alone, however, does not release these factors.

Therefore, these enzymes and substrates are considered to be in solution in the matrix in the compartment enclosed by the inner membrane. The single exception is succinic acid dehydrogenase, which, with its prosthetic group (FAD), forms a part of the electron transport system and is tightly bound to the cristae membranes as an integral membrane protein.

Enzymatic activity of the outer mitochondrial membrane is relatively limited. One enzyme, associated with the oxidation of amino acids, an amine oxidase, is so characteristic of the outer mitochondrial membrane that it is used as a marker to identify this membrane fraction in isolated preparations. Other enzymes have also been identified within the outer membranes, including the complex oxidising pyruvic acid.

Given the extreme complexity and variety of the enzyme systems present in mitochondria, which, in addition to oxidative enzymes, also include full facilities for DNA and protein synthesis, these organelles are probably the most biochemically complicated and capable structures inside animal cells. In plant cells they are rivaled only by chloroplasts, are equally diverse in their bioc­hemical activity.

Both fats and proteins, can serve as cellular fuels. As a preliminary to their cellular oxidation, these comparatively large molecules are first broken into smaller units- Fats into glycerol and fatty acids and proteins into amino acids. The smaller units are then oxidised in reactions that take place primarily inside mitochondria. The individual amino acids derived from hydrolysis of proteins are oxidised in a variety of pathways.

These pathways, depending on the particular amino acid, eventually yield products that enter the main line of carbohydrate oxidation either as pyruvic acid, acetyl coenzyme A, or intermediates of the Krebs cycle. As a part of the pathways leading to these entry points, the amino acids are deaminated (the —NH₂ group is removed).

Products of both fat and protein breakdown thus enter the central carbohydrate pathway to be oxidised to CO₂ and H₂O primarily in mitochondria summarises these routes. The central role played by coenzyme A as a carrier funneling products of different pathways into the Kerbs cycle.

The end product of all of these reactions that is significant to cellular activity is ATP. Additional oxidative pathways of importance are summarised in the supplements. Supplement describes the pentose phosphate pathway, an alternate route for carbohydrate oxidation that also serves as a source of five-carbon sugars. Additional pathways for the oxidation of fats and amino acids, taking place in microbodies (peroxisomes and glyoxisomes).

Integration of Mitochondrial Activity with the Cell Environment:

Mitochondrial Transport:

The reaction pathways of pyruvic acid oxidation, fatty acid Dzidation, and the Krebs cycle are located inside the mitochondrial matrix. How are the substrates for these reactions and the ADP-ATP couple transported in and out of mitochondria? The outer mitochondrial membrane is freely permeable to most organic and inorganic molecules with molecular weights up to about 5000. Both mitochondrial membranes freely admit H₂O, O₂, and CO₂.

However, the inner mitochondrial membrane surrounding the matrix is almost completely impermeable to most hydrophilic substances, except for a group of molecules that are transported by specific carrier systems.

Several of these systems have been identified reviewed in LaNoue and Schoolwerth. One, called the adenine nucleotide carrier, exchanges ATP for ADP in either direction across the inner mitochondrial membrane.

Another carrier mechanism, shuttles electrons carried by reduced NAD outside to NAD inside the mitochondrion. Other specific carriers transport intermediates of the various oxidative pathways located in the matrix, including inorganic phosphate, fatty acid derivatives, pyruvic acid, various amino acids, and acids of the Kerbs cycle.

Various lines of evidence indicate that the inner membrane carriers are proteins. They are highly specific for the molecules or molecular groups carried and exhibit saturation at high concentrations of the transported substances.

Some of the carrier proteins, including the adenine nucleotide carrier exchanging ATP for ADP across the inner membrane have been isolated and partially purified. Addition of the nucleotide carrier protein to artificial phospholipid bilayers transfers limited ATP-ADP transport capability to the bilayers.

Most of the transport systems of the inner membrane are active, and thus require energy for their functions. In mitochondria, the carriers are evidently driven by the H⁺ gradient set up across the inner membrane by electron transport.

The electron transport system activity expels H⁺ ions into the inter-membrane compartment, setting up the H⁺ gradient. Diffusion of the H⁺ ions back into the matrix is tightly coupled to the inward transport of the substances admitted by the inner membrane carriers. Transport of these substances thus occurs by the H⁺-linked co-transport pathway.

Regulation of Mitochondrial Oxidation:

The rate of mitochondrial oxidation is regulated by a system of controls within the cell. Of these, the most important is based on the concentration of ADP in the medium surrounding the mitochondrion. As ADP concentration increases, phosphorylation of ADP to ATP begins inside mitochondria. This phosphorylation is closely coupled to electron transport; as phosphorylation takes place, electrons flow without restriction from NAD to oxygen, and the NAD end of the transport chain becomes relatively oxidised.

NAD, once oxidised, is free to recycle as an electron acceptor in reactions oxidising the substrates of the Krebs cycle. This system closely links the rate of oxidation in the Krebs cycle to the concentration of ADP in the cell.

If cellular activity is limited, ATP concentration will remain high, and ADP will become unavailable to the ATP-synthesizing enzyme complex in cristae membranes. As a result, the ATP-synthesizing reactions and electron transport will stop.

According to the Mitchell hypothesis, stoppage of electron transport is due to a buildup in the H⁺ gradient that is unrelieved by ATP synthesis. The gradient gradually builds until it reaches levels high enough to oppose further expulsion of H⁺ across the cristae membranes by the electron transport system. In consequence, electron transport and, in turn, oxidation in the Krebs cycle stop.

Control of glycolysis may also be linked to mitochondrial activity through the oxidation-reduction cycle of extra mitochondrial NAD. Continuance of glycolysis depends on re-oxidation of the NAD reduced in the sequence.

Through the NAD shuttle systems, extra mitochondrial reduced NAD may transfer its electrons to NAD or FAD inside the mitochondrion to become re-oxidised and cycle back to glycolysis. Passage of electrons through the shuttle systems depends on the availability of oxidised NAD or FAD inside the mitochondrion.

This in turn depends on the rate of electron transport and ADP concentration. As cells carry out their major activities, including growth, movement, and response to their environment, ATP is hydrolysed to ADP.

The increase in ADP concentration causes an increase in the rate of oxidation in the mitochondrion, and the concentration of ATP is restored. All the various activities, from glycolysis through synthesis, thus exists as a delicately balanced system, in which the Oxidative