In this article we will learn about the formation of ATP in the respirator chain.
Advantage of an Oxidation by Steps:
Glucose or glucosyl equivalents, are our main source of energy; we will take them as example to illustrate the relations between cellular oxidations and ATP formation. But it must be said that other compounds can be a source of energy for living organisms that triglycerides have (for equal weight) a greater energetic value than carbohydrates.
We have already stated that the complete oxidation of glucose (e.g., by combustion in air) according to the reaction C6H12O6 + 6O2 → 6CO2 + 6H2O liberates 686 kcal/mole.
If we compare this value to the energy required for the synthesis of ATP by the reaction ADP + Pi → ATP + H2O (8 to 10 kcal/mole on an average, in the usual intracellular conditions), it appears that theoretically, the oxidation of one mole of glucose can yield sufficient energy for the synthesis of 686/9 # 76 moles of ATP.
The complete oxidation of glucose into CO2 + H2O in the usual pathway (glycolysis followed by the Krebs cycle and the respiratory chain) permits the synthesis of 38 ATP per mole of glucose (i.e. half the theoretical figure), and the rest of the energy is liberated as heat; this may be summarized as follows:
This is actually a diagram which summarizes the whole set of reactions, and not a single reaction because, if there had been only one reaction, only one molecule of ATP would be formed and all the remaining energy would be dissipated in the form of heat. The oxidation of glucose takes place in successive steps, with liberation of a small fraction of the total energy each time, thus enabling the formation of several molecules of ATP.
Anticipating to some extent, it may be said that glucose is oxidised in 6 steps: one oxidation step in the glycolysis (reaction 4, fig. 4-35), one in the decarboxylation of pyruvic acid (see fig 4-36), and four in the Krebs cycle (reactions 3, 4, 6 and 8, fig. 4-38).
But one could reasonably wonder how 38 molecules of ATP can be produced during 6 oxidation reactions only, because on examining one of these reactions, for example, the dehydrogenation (or oxidation) of isocitric acid to oxalo-succinic acid (see fig. 3-4), it is observed that isocitric acid loses, to the advantage of oxygen, 2 hydrogen atoms and 2 electrons (since oxygen passes from oxidation state zero to state -2); and although this is the minimum oxidation which isocitric acid can undergo, it would not be very economic from the energetic point of view, if it look place in a single step, because only one molecule of ATP could be formed out of the 5 which could arise thanks to the 50 kcal made available (and some 40 kcal which remain would be dissipated as heat).
In fact, for each molecule of isocitric acid oxidized into oxalo-succinic acid, 3 molecules of ATP (not one) are formed. The reaction — as it is written above — does not reflect the real situation because the electrons do not pass directly from isocitric acid to oxygen. As will be seen in the following, a series of oxidation-reduction reactions enable a transfer of electrons by steps, and three of them are coupled with the formation of one molecule of ATP.
Electron Carrier System or Respiratory Chain:
In certain cases, the electrons of the substance to be oxidized pass directly to oxygen, but in general, electrons are transferred in successive steps to a series of carriers, which is much more advantageous from the energetic point of view. One must also note that the sudden oxidation of substances like glucose with liberation of 686 kcal/mole, could not take place in living organisms, because it would be accompanied by an excessive release of heat as only one ATP can form per reaction.
Furthermore — and this is another advantage — the electrons originating from the oxidation of very diverse substrates are channelled towards the same electron carrier system to finally reach oxygen. This system can vary slightly from one organism to another, but in general it contains: an enzyme (dehydrogenase) having as coenzyme a pyridine nucleotide (NAD or NADP), a flavoprotein, coenzyme Q and several cytochromes.
These compounds represent in mitochondria about 1/4 of total proteins; they are grouped in multi-enzymatic aggregates forming complex structures called electron carrier particles; this facilitates the transfer of electrons and increases the efficiency of this oxidation chain whose role is to enable the oxidation of various compounds with reduction of oxygen to H2O and formation of ATP. This respiration — or enzymatic oxidation of nutritive substances by molecular oxygen — is the principal source of energy of aerobic cells.
A. The Electron Carriers:
These carriers must be considered as being initially in the oxidized state; they will be reduced by hydrogen or by electrons originating either from the substrate or from the previous carrier; then, they will be re-oxidized by the next carrier. These are coupled reactions, of catalytic nature: the various carriers can thus be reoxidized after having functioned and they can hence serve a large number of times; oxidations can therefore be carried out by relatively small quantities of the various carriers.
a) NAD or NADP Dehydrogenases:
The structures of the oxidized and reduced forms of NAD, the NAD+ or (NADP+) can bind 2 electrons and a hydrogen atom removed from a substrate (which is thus oxidized), and be reduced to NADH + H+ (or NADPH + H+).
A wide variety of substances are oxidized in this manner: glyceraldehyde-3-phosphate (see fig. 4-27), isocitric acid, malic acid (see fig. 4-38), glucose-6-phosphate (see fig. 4-39), the hydroxy-acyl-co A during the β-oxidation of fatty acids (fig. 5-11), etc.
b) Flavoproteins:
The structures of coenzymes derived from riboflavin (FAD, FMN) and the manner in which they participate in oxidation-reduction processes. In the respiratory chain, the flavoproteins allow the reoxidation of NADH to NAD+ and upon binding a hydrogen atom of NADH, a H+ of the solution and 2 electrons of NADH, they are then in the reduced state. We will see (figs. 3-6 b and 4-38) that in certain cases a flavoprotein can bring about the oxidation of a substrate like succinate (without passing through NAD).
c) Coenzyme Q:
The reduced flavins can be reoxidized by transfer of 2 hydrogen atoms (2H+ and 2e–) to a molecule of coenzyme Q which is then in a reduced state. The structure of this compound is shown in figure 5-9; it is a substituted benzoquinone having an isoprenic side chain, which is related to that found in some lipids.
The number of isoprene units in this chain can vary according to the origin of the compound. The coenzyme Q10 (10 isoprene units) also called ubiquinone 50 (10 units of 5 carbon atoms = 50 carbon atoms) is the most common form in animal mitochondria. Like quinones in general, the coenzyme Q can undergo a reversible reduction to hydroquinone (see fig. 3-5). The coenzyme Q or ubiquinone is a soluble mobile carrier in the lipidic bi-layer of the mitochondrial inner membrane.
Flavoproteins like the coenzymes Q ensure two successive transfers of 1H + and 1e–.
d) Cytochromes:
As indicated by their name, these are coloured substances. Cytochromes are chromoproteins, the prosthetic group of which — strongly linked to the protein — is a porphyrin, identical or very similar to the heme of hemoglobin (see fig. 1-29). The various cytochromes — 5 of them are known to belong to the mitochondrial system of electron transport — differ either in the protein part, or in the linkages between this protein part and the porphyrin, or in certain cases by the substituents on the β-carbort atoms of the pyrrole rings of porphyrin.
As observed below (see Table above), these structure differences result in differences in reactivity and especially in the oxidation-reduction potential. Contrary to the former carriers, cytochromes can transport only one electron at a time and that is why 2 molecules of each cytochrome are involved in the reactions of the respiratory chain in which they participate (see fig. 3-6).
The first to participate in the respiratory chain is cytochrome b which enables the re-oxidation of coenzyme Q. It must be noted that at this stage, the 2 hydrogen atoms are not transferred, but 2 protons are liberated in the medium. Since the reduction of one molecule of cytochrome b implies the transfer of only one electron, the participation of 2 molecules of cytochrome b is needed to accept the 2 electrons and allow the re-oxidation of the coenzyme Q. It is important to note that the reduced form of cytochromes contains the ferrous ion Fe2+, while the oxidized form has a ferric ion Fe3+ (whereas the hemoglobin ←→ oxyhemoglobin transformation takes place without any change of the state of oxidation of iron).
The electrons pass from cytochrome b to cytochrome c1, then to cytochromes c, a, a3 by the same process of oxidation and reduction of iron. Finally, the electrons reach the cytochrome a3 also called cytochrome-oxidase which has the peculiarity of being directly oxidizable by molecular oxygen, reaction in which participate more than 9/10th of the oxygen utilized by the cells.
This enzyme is inhibited by small concentrations of cyanide (CN–), so that intoxication by this ion inhibits electron transport and synthesis of ATP by the respiratory chain, which generally results in rapid death.
This last reaction of the chain — contrary to the previous reactions — is irreversible, which suffices to drag the electron carrier system in the direction in which it usually functions: from the oxidizable substrate to the oxygen (from top to bottom in the diagram of figure 3-6 a).
B. Oxidation-Reduction Potentials:
If an electrode of un-attackable metal (e.g., Pt) is dipped in a solution of a reducing substance A (i.e. a substance which can yield electrons), the latter will yield electrons to the metal which will thus get negatively charged. If on the contrary, the metal electrode is placed in a solution containing an oxidizing substance B (which can accept electrons), the latter will extract electrons from the metal which will then be positively charged.
If the 2 solutions are connected by a conducting bridge and the 2 electrodes by a voltmeter, one can observe on the latter the flow of an electric current through the wire due to the transfer of electrons from the electrode (-) to the electrode (+).
This is the principle of the electric cell, where the potential difference is very large at the start if A is highly reducing and B highly oxidizing, but decreases progressively as A is oxidized and B is reduced, and finally becomes nil; at each instant this potential difference is determined by the respective proportions of oxidized and reduced substance in each of the two compartments.
Let us now consider one compartment only: depending on whether there is more of A reduced or A oxidized, the electrode will be charged negatively or positively (if the concentrations of the 2 forms are equal, the charge is nil). If we connect this electrode to a reference electrode, generally the normal hydrogen electrode (a Pt blade covered with Pt black saturated with gaseous hydrogen, dipping in a solution containing one ion-gram hydrogen per litre), whose potential is, by definition, equal to 0, we will measure a potential difference (E), representing the oxidation-reduction potential of the system. Such an apparatus is shown in fig. 3-7: on the left we have the reaction 2H ←→ 2H + + 2e–, which will make the Pt electrode negatively charged; on the right, we will have the reaction: 2A (oxidized form) + 2e– (pulled out of the electrode) ←→ 2A (reduced form), so that the electrode will be positively charged.
The potential difference is given by the following relation (Nernst formula):
where E is the potential difference (in volts or millivolts) between the electrode of the oxidation-reduction system under study and the reference electrode,
E0 is the normal oxidation-reduction potential, i.e. the potential difference measured between the normal hydrogen electrode and the electrode dipping in the oxidizing-reducing system containing as much oxidized A as reduced A.
R is the gas constant (1.987 cal/mole/degree), T the absolute temperature, n the number of electrons transferred per mole and 
the faraday (96 500 coulombs). As for [oxidized form] and [reduced form], they are the activities of the oxidized and reduced forms which can be – on a first approximation — taken as the molar concentrations.
If these 2 concentrations are equal, their ratio is 1, and since the log of 1 is 0, we have in this case E = E0. It is clear that variations of the respective concentrations of the oxidized and reduced forms will result in modifications of the value of the oxidation-reduction potential.
One must note the resemblance between the above relation and the corrected formula giving the free energy change. The oxidation- reduction potential represents in fact one way of measuring the free energy of a reduction reaction. We will see in the following, the exact relation between ∆E and ∆G, and how this relation can help in determining the stages in which ATP is formed.
It was found that for biochemical oxidation-reductions, the conditions defined for the normal hydrogen electrode, i.e. 25°C, hydrogen pressure = 1 atmosphere, [H+] = 1 (i.e. pH = 0), were not very physiological (particularly the latter); a normal potential E’0 corresponding to 30°C and pH 7 (which replaces E0 in Nernst formula) was therefore defined. In these conditions, the potential of the normal hydrogen electrode is not 0, but E’0 = – 0.42 volt.
When there are several oxidation-reduction systems (or redox systems) in the same series of reactions, it is of interest to know their oxidation-reduction potentials.
For example, when there are 2 systems:
A oxidized + e– → A reduced (with potential EA)
B oxidized + e– → B reduced (with potential EB)
Let us suppose EA > EB, i.e. that system A is more oxidizing than system B; this means that if the 2 systems are in contact, the electrons will tend to pass from B to A, in other words B will get oxidized while A will be reduced. Electrons tend to pass from the earner having the most negative potential to the carriers having less negative or increasingly positive potentials.
The oxidation-reduction potentials of a large number of biological oxidation-reduction systems were determined (with respect to a reference system), notably, the potentials of carriers of the respiratory chain which are of special interest to us; the values are given in the table.
This table, showing the values of potential differences of oxidation-reduction at pH 7 and 30°C (AE’0), also contains the values (∆G’0) of standard free energy changes at pH 7 and 30°C which enable us to locate the stages of phosphorylation. The ∆G’ values differ depending on the general equilibrium of the respiratory chain, but the effective potential jumps remain the same.
It can be seen that the oxidation-reduction potentials of the various systems increases continuously from pyridine nucleotide enzymes to oxygen; this indicates the theoretical order in which the transfer of electrons must take place (we mentioned above that the transfer of electrons takes place in the direction of a system having a higher potential); this theoretical order is indeed the same as the order in which the electron carriers of the respiratory chain follow one another (see fig. 3-6). It must be noted that the oxidation-reduction potentials of coenzymes (indicated below) can be modified when the coenzyme is linked to the apoenzyme.
C. Phosphorylations in the Respiratory Chain:
The free energy change between 2 redox systems (∆G’0) is related to the difference between the oxidation-reduction potentials of the 2 systems (∆E’0) by the following equation:
where n is the number of electrons transferred and F the Faraday; but the use of one Faraday per mole of substrate for a valency modification of one unit, produces an energy of 23.06 kcal/volt; the Faraday is therefore equivalent to 23.06 kcal/volt.
If one considers the normal potential difference of oxidation- reduction between the beginning (NAD) and the end (oxygen) of the respiratory chain (1.13 volts), one can — with the help of the above equation — calculate that the oxidation of the H+ and e– of NADH + H+ can supply energy amounting to:
∆G’0 = 2 X 23.06 X 1.13 # -52 kcal
We know that this energy is not liberated in one stroke but in successive steps. The problem is to know which steps will lead to the formation of ATP. Since the formation of ATP from ADP + P, + H+ requires about 7 kcal, it may be said that those steps of the respiratory chain in which the energy liberated is greater than 7 kcal, should enable the synthesis of ATP. It is obvious that ∆G’ for the synthesis or hydrolysis of ATP depends on pH since H+ is involved.
If we write:
we can deduce ∆E’0 = 7/2 X 23.06 = 0.152 volt; in other words, to a normal potential difference greater than 0.152 volt corresponds an energetic variation sufficient for the formation of one molecule of ATP. The table contains the ∆E’0 and ∆G’0 values corresponding to each step, and it may be observed that 3 steps should lead to the synthesis of ATP: the passage of electrons from the pyridine coenzyme to the flavin coenzyme, from cytochrome b to cytochrome c, and from cytochrome a to oxygen.
In these 3 steps there is possibility of coupling between oxidation-reduction and phosphorylation of ADP to ATP, hence the name oxidative phosphorylations or phosphorylating oxidation-reductions given to these coupled reactions in the respiratory chain.
The above reasoning is based on normal oxidation-reduction potentials (E’0) for the various electron carriers and on standard free energy changes (∆G’0), but it is certain that these values must be corrected in order to reflect the intracellular (or more exactly the intra-mitochondrial) conditions. Furthermore, these are theoretical predictions and it remains to be seen whether they have been confirmed experimentally.
If mitochondria are incubated in a suitable medium, in presence of an oxidizable substrate, oxygen, ADP and Pi, one can measure the disappearance of the substrate, ADP or Pi, the consumption of oxygen and the formation of ATP.
One can thus define, for the oxidation of each substrate, a phosphorylation ratio (P/O), i.e. the number of molecules of ATP formed (or molecules of Pi consumed) per atom of oxygen utilized (or per molecule of hydrogen removed from the substrate).
This P/O ratio is equal to 3 when the acceptor is a pyridine coenzyme like NAD+, as for example in the case of the oxidation of isocitric acid or malic acid in the Krebs cycle (see fig. 4-38). But the P/O ratio is only 2 during another oxidation of the Krebs cycle, that of succinic acid, because in this reaction the acceptor is FAD, so that one oxidation-reduction step coupled with a phosphorylation is skipped and only 2 phosphorylating steps remain.
The ratio can even be only one when the acceptor is cytochrome c, for example, in the case of the oxidation of ascorbic acid, because 2 steps of phosphorylating oxidation are skipped. On the contrary, the ratio can also be greater than 3; the P/O ratio is 4 for example for the oxidation of α-ketoglutaric acid to succinic acid, because the GTP formed leads to the formation of one additional molecule of ATP (see fig. 4-38).
Experimental results have therefore confirmed the theoretical predictions which suggested that three steps of the respiratory chain are coupled with the formation of ATP.
However, this coupling is not absolutely obligatory and there are substances which can uncouple the phosphorylating oxidations (thyroxine which is a hormone secreted by thyroid, 2, 4-dinitrophenol, etc.): oxidations continue, but oxidative phosphorylations are inhibited so that the energy, instead of being utilized for the formation of ATP, is dissipated as heat, which brings about a temperature rise in the animal that has been administered an uncoupling substance. However, the phosphorylations bound to the substrate are not affected by the uncoupling agents.
D. The Shuttles:
Mitochondria possess a folded inner membrane (forming crests), with a very large functional surface (see fig. 3-8). The major oxidation-reduction processes take place in the inner membrane or on the inner face (matrix side) of the inner membrane, and it is in this inner membrane that are found the lipoic acid dehydrogenases of pyruvate and α-ketoglutarate, other dehydrogenases like succinodehydrogenase, enzymes of the Krebs cycle and of β-oxidation of fatty acids, the carriers participating in the respiratory chain and the system of synthesis of ATP coupled with the respiratory chain. 
The electron carriers of the respiratory chain drain the maximum of protons and electrons originating from the dehydrogenations taking place either in the mitochondrion (notably in the inner membrane), or in the cytosol. But the inner membrane has a selective permeability and nucleotides of the type NAD(P) for example, cannot pass through it, so that the electrons and protons from the cytosol are used to temporarily reduce a substrate, like oxaloacetate, into malate, which can cross the inner membrane thanks to a carrier and can then be reoxidized (to oxaloacetate) yielding NADH + H+ which will enter the respiratory chain. This is an example of shuttle, enabling the passage of the reducing equivalents from the cytosol to the mitochondrion (see fig. 3-9).
As mentioned above, the substrates originating from the cytoplasm must use specialized carriers (or translocators) to penetrate into the mitochondrion and be either oxidized or phosphorylated (in the case of ADP and phosphate). One knows for example, carriers of dicarboxylic acids (malate, succinate), tricarboxylic acids (citrate, isocitrate), fatty acids, α-ketoglutarate, glutamate, phosphate, ADP and ATP.
These carriers can be selectively inhibited by certain substances, blocking the passage of substrates (and therefore of protons and electrons) through the inner membrane of mitochondria and thereby inhibiting the oxidation-reductions of the respiratory chain.
But cellular oxidation-reductions can also be affected by inhibitors of oxidative decarboxylations (pyruvate, α-ketoglutarate) or of the Krebs cycle, by perturbation of oxygen uptake (especially by substances affecting hemoglobin, like CO), by inhibitors of electron carriers of the respiratory chain (some are indicated in fig. 3-6 b), by uncoupling agents which dissociate the synthesis of ATP from the oxidations of the respiratory chain so that the substrates are oxidized in pure loss with production of heat (by 2,4-denitrophenol, thyroxine, for example), or by inhibitors of the synthesis of ATP (like oligomycin).
E. Energy Yield of the Oxidation of Glucose in Aerobic Cells:
At the beginning of this study devoted to the formation of ATP during oxidation processes, glucose was taken as example. The complete oxidation of one molecule of glucose, through glycolysis followed by the Krebs cycle, allows the formation of 38 molecules of ATP which corresponds to about 38 x 7 = 266 kcal/mole of glucose. But it is known that the sudden combustion of glucose could yield 686 kcal/mole.
It is therefore easy to calculate the efficiency of energy conservation: 266 x 100/686 # 40%. This is a rather remarkable yield, but it should be noted that this value is obtained from ∆G0 (valid in standard conditions) and that if one considered the real intracellular concentrations in ADP, Pi, and ATP, the yield obtained would be still higher. Indeed, this yield is 50% if one considers that on average, 9 kcal are required to form one molecule of ATP in the intracellular conditions.
F. Regulation of the Oxidative Phosphorylation:
The mitochondrion can be regarded as a small transformation factory which receives from the cellular cytoplasm, substrates to be oxidized, oxygen, ADP and Pi, and produces CO2, water and ATP. One might think that the oxidation velocity of a substrate like an acetate equivalent by the Krebs cycle and the respiratory chain depends mainly on concentrations of acetyl-coA and oxygen.
In reality it mostly depends on the respective concentrations of ATP, ADP and Pi and the ATP/ADP ratio has a decisive influence on respiration.
For example, in a resting muscle, where the utilization of ATP to give ADP + Pi (reaction required for muscle contraction) is low, the ATP/ADP ratio is comparatively high and the rate of respiration is low, because respiration needs ADP (without ADP, no phosphorylating oxidation); on the contrary, in a muscle at work, the decomposition of ATP into ADP in ADP + Pi is considerable, the ATP/ADP ratio therefore decreases, and the fact that ADP and P, are available in larger quantities permits an acceleration of respiration (and consequently, a regeneration of the ATP required for muscle work).
This regulation by the ATP/ADP ratio obviously assumes that the substrate (here acetyl-coA) and oxygen are in excess in the cell; when these conditions are fulfilled (and they generally are) the production of A TP by oxidative phosphorylation is therefore controlled by the A TP requirements of the cell, whether for muscle contraction, to allow endergonic reactions, or for an altogether different purpose (see fig. 3-3).
