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Theory Notes

Which of the following occurs in non shivering thermogenesis?

A) Glucose is oxidized to lactate

B) Fatty acids uncouple oxidative phosphorylation

C) ATP is spent for heat production

D) Glycogen is excessively degraded

E) Fatty acids are excessively oxidized

The correct answer is C- Fatty acids uncouple oxidative phosphorylation

Basic Concept

The reduction of molecular oxygen to water yields a large amount of free energy that can be used to generate ATP. Oxidative phosphorylation is the process in which ATP is formed as a result of the transfer of electrons from NADH or FADH2 to O2 by a series of electron carriers (figure-1).This process, which takes place in mitochondria, is the major source of ATP in aerobic organisms.

Electron transport chain

Figure-1-Flow of electrons in the electron transport chain and ATP synthase complex. The flow of electrons from NADH or FADH2 to O2 through protein complexes located in the mitochondrial inner membrane leads to the pumping of protons out of the mitochondrial matrix. The resulting uneven distribution of protons generates a pH gradient and a transmembrane electrical potential that creates a proton-motive force. ATP is synthesized when protons flow back to the mitochondrial matrix through an enzyme complex. In other words, the electron-motive force is converted into a proton-motive force and, finally, the proton-motive force is converted into phosphoryl transfer potential.

Thus, the oxidative phosphorylation or in other words, oxidation of fuels and the phosphorylation of ADP are coupled processes.

Uncouplers of oxidative phosphorylation

This tight coupling of electron transport and phosphorylation in mitochondria can be disrupted (uncoupled) by

  1. 2,4- Dinitrophenol ,
  2. 2,4  dinitrocresol
  3. CCCP( chloro carbonyl cyanide phenyl hydrazone)
  4. FCCP
  5. Valinomycin
  6. High dose of Aspirin
  7. The antibiotic Oligomycin completely blocks oxidation and phosphorylation by blocking the flow of protons through ATP synthase complex.

These substances carry protons across the inner mitochondrial membrane. In the presence of these uncouplers, electron transport from NADH to O2 proceeds in a normal fashion, but ATP is not formed by mitochondrial ATP synthase because the proton-motive force across the inner mitochondrial membrane is dissipated. This loss of respiratory control leads to increased oxygen consumption and oxidation of NADH. Indeed, in the accidental ingestion of uncouplers, large amounts of metabolic fuels are consumed, but no energy is stored as ATP. Rather, energy is released as heat.

Physiological uncouplers

1) Long chain fatty acids

2) Thyroxin

3) Brown Adipose tissue-Thermogenin (or the uncoupling protein) is a physiological uncoupler found in brown adipose tissue that functions to generate body heat, particularly for the newborn and during hibernation in animals

4) Calcium ions.

The regulated uncoupling of oxidative phosphorylation is a biologically useful means of generating heat. The uncoupling of oxidative phosphorylation is a means of generating heat to maintain body temperature in hibernating animals, in some newborn animals (including human beings), and in mammals adapted to cold. Brown adipose tissue, which is very rich in mitochondria (often referred to as brown fat mitochondria), is specialized for this process of non shivering thermogenesis. The inner mitochondrial membrane of these mitochondria contains a large amount of uncoupling protein (UCP), here UCP-1, or Thermogenin, a dimer of 33-kd subunits that resembles ATP-ADP translocase. UCP-1 forms a pathway for the flow of protons from the cytosol to the matrix. In essence, UCP-1 generates heat by short-circuiting the mitochondrial proton battery. This UCP-1 channel is activated by fatty acids (as in the given case) – Figure-2.

Brown adipose tissue

Figure-2- Process of uncoupling of oxidative phosphorylation in the Brown adipose tissue.

Thus the most appropriate answer in the given situation is – uncoupling of oxidative phosphorylation by fatty acids.

As regards other options

A) Glucose is oxidized to lactate- under anaerobic conditions or in the cells lacking mitochondria. The released energy is captured as 2 mols of ATP; the surplus energy is released as heat to maintain body temperature.

C) ATP is never spent for heat production

D) Glycogen is not excessively degraded in non shivering thermogenesis

E) Fatty acids are not excessively oxidized to yield extra energy as heat.

The manner in which biologic oxidative processes allow the free energy resulting from the oxidation of foodstuffs to become available and to be captured is stepwise, efficient, and controlled—rather than explosive, inefficient, and uncontrolled, as in many non biologic processes. The remaining free energy that is not captured as high-energy phosphate is liberated as heat. This need not be considered “wasted,” since it ensures that the respiratory system as a whole is sufficiently exergonic, allowing continuous unidirectional flow and constant provision of ATP. It also contributes to maintenance of body temperature.

 

 

 

 

 

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ATP SYNTHASE COMPLEX

 ATP synthase is embedded in the inner membrane, together with the respiratory chain complexes .

  • Several subunits of the protein form a ball-like shape arranged around an axis known as F1, which projects into the matrix and contains the phosphorylation mechanism .
  • F1 is attached to a membrane protein complex known as F0, which also consists of several protein subunits (Figure-1).
  • F0 spans the membrane and forms a proton channel.
  • The flow of protons through F0 causes it to rotate, driving the production of ATP in the F1 complex.

 ATP Synthase complex

Figure-1-The enzyme complex consists of an F0 sub complex which is a disk of “C” protein sub units. Attached is a Υ subunit in the form of a “bent axle.” Protons passing through the disk of “C” units cause it and the attached Υ subunit to rotate. The Υ subunit fits inside the F1 sub complex of three α and three β subunits, which are fixed to the membrane and do not rotate.

OXIDATIVE PHOSPHORYLATION

Chemiosmosis

  • As the electrons are transferred, some electron energy is lost with each transfer.
  • This energy is used to pump protons (H+) across the membrane from the matrix to the inner membrane space. A proton gradient is established (Figure-2)
  • The higher negative charge in the matrix attracts the protons (H+) back from the intermembrane space to the matrix.
  • The accumulation of protons in the intermembrane space drives protons into the matrix via diffusion.
  • Most protons move back to the matrix through ATP synthase.
  • ATP synthase uses the energy of the proton gradient to synthesize ATP from ADP + Pi

 

 Proton motive force

Figure-2- Showing electrochemical gradient across the inner mitochondrial membrane. The chemiosmotic theory, proposed by Peter Mitchell in 1961, postulates that the two processes are coupled by a proton gradient across the inner mitochondrial membrane so that the proton motive force caused by the electrochemical potential difference (negative on the matrix side) drives the mechanism of ATP synthesis.

 P: O Ratio

  •  Defined as the number of inorganic phosphate molecules incorporated in to ATP for every atom of oxygen consumed.
  • Oxidation of NADH yields 3 ATP molecules(P: O ratio 3, Latest concept 2.5)
  • Oxidation of FADH2 yields 2 ATP molecules (P: O ratio 2, Latest concept 1.5)

Inhibition of Oxidative phosphorylation

Oxidative phosphorylation is susceptible to inhibition at all stages of the process.  

A) Site specific inhibitors -Specific inhibitors of electron transport chain are (figure-3) for example-

1) Inhibitors of complex IRotenone and amobarbital block electron transfer in NADH-Q oxidoreductase and thereby prevent the utilization of NADH as a substrate. In contrast, electron flow resulting from the oxidation of succinate is unimpaired, because these electrons enter through QH2, beyond the block. The other inhibitors are  ChlorpromazinePiericidin A and Guanethidine

2) Inhibitors of Complex II

Malonate is a competitive inhibitor of Complex II. The other inhibitors are Carboxin and TTFA

 Site specific inhibitors of ETC

Figure-3- Site specific inhibitors block the flow of electron at specific sites

3) Inhibitors of Complex III

BAL (British Anti Lewisite), Antimycin A, Naphthoquinone  and hypoglycemic agents  interfere with electron flow from cytochrome b in Q-cytochrome c oxidoreductase.

4) Inhibitors of Complex IV

Furthermore, electron flow in cytochrome c oxidase can be blocked by hydrogen sulphide (H2S),  cyanide (CN-),azide (N3 -), and carbon monoxide (CO). Cyanide and azide react with the ferric form of heme a 3, whereas carbon monoxide inhibits the ferrous form. Inhibition of the electron-transport chain also inhibits ATP synthesis because the proton-motive force can no longer be generated.

B) Inhibitors of ATP synthase complex 

Oligomycin and dicyclohexyl carbodiimide(DCCD) prevent the influx of protons through ATP synthase. If actively respiring mitochondria are exposed to an inhibitor of ATP synthase, the electron transport chain ceases to operate. Indeed, this observation clearly illustrates that electron transport and ATP synthesis are normally tightly coupled.

C) Inhibition of ATP-ADP translocase

ATP-ADP translocase is specifically inhibited by very low concentrations of Atractyloside (a plant glycoside) or Bongregate (an antibiotic from a mold).Unavailability of ADP also inhibits the process of ATP formation. This is because oxidation and phosphorylation are tightly coupled; ie, oxidation cannot proceed via the respiratory chain without concomitant phosphorylation of ADP.

 ADP-ATP Transport system

Figure-4- ADP moves in to the mitochondrial matrix and newly synthesized ATP is transported out in to the cytoplasm to be used for cellular processes

D) Uncouplers of Oxidative phosphorylation

The tight coupling of electron transport and phosphorylation in mitochondria can be disrupted (uncoupled) by agents called uncouplers of oxidative phosphorylation. These substances carry protons across the inner mitochondrial membrane. In the presence of these uncouplers, electron transport from NADH to O2 proceeds in a normal fashion, but ATP is not formed by mitochondrial ATP synthase because the proton-motive force across the inner mitochondrial membrane is dissipated. This loss of respiratory control leads to increased oxygen consumption and oxidation of NADH. Indeed, in the accidental ingestion of uncouplers, large amounts of metabolic fuels are consumed, but no energy is stored as ATP. Rather, energy is released as  heat.

Examples

Physiological Uncouplers

  • Long chain fatty acids
  • Thyroxin
  • Brown Adipose tissue-Thermogenin (or the uncoupling protein) is a physiological uncoupler found in brown adipose tissue that functions to generate body heat, particularly for the newborn and during hibernation in animals
  • Calcium ions.

The regulated uncoupling of oxidative phosphorylation is a biologically useful means of generating heat. The uncoupling of oxidative phosphorylation is a means of generating heat to maintain body temperature in hibernating animals, in some newborn animals (including human beings), and in mammals adapted to cold. Brown adipose tissue, which is very rich in mitochondria (often referred to as brown fat mitochondria), is specialized for this process of non shivering thermogenesis. The inner mitochondrial membrane of these mitochondria contains a large amount of uncoupling protein (UCP), here UCP-1, or thermogenin,a dimer of 33-kd subunits that resembles ATP-ADP translocase. UCP-1 forms a pathway for the flow of protons from the cytosol to the matrix. In essence, UCP-1 generates heat by short-circuiting the mitochondrial proton battery(figure-5).

Thus, the manner in which biologic oxidative processes allow the free energy resulting from the oxidation of foodstuffs to become available and to be captured is stepwise, efficient, and controlled—rather than explosive, inefficient, and uncontrolled, as in many non biologic processes. The remaining free energy that is not captured as high-energy phosphate is liberated as heat. This need not be considered “wasted,” since it ensures that the respiratory system as a whole is sufficiently exergonic, allowing continuous unidirectional flow and constant provision of ATP. It also contributes to maintenance of body temperature.

 

Uncoupling of oxidative phosphorylation 

Figure-5- UCP-1 forms a pathway for the flow of protons from the cytosol to the matrix. The proton gradient is dissipated, oxidation proceeds without phosphorylation.

Pathological uncouplers

These compounds are toxic in vivo, causing respiration to become uncontrolled, since the rate is no longer limited by the concentration of ADP or Pi.

  •  2,4-dinitrophenol
  • 2, 4- dinitrocresol
  • CCCP( chloro carbonyl cyanide phenyl hydrazone)
  • FCCP
  • Valinomycin
  • High dose of Aspirin
  • The antibiotic Oligomycin completely blocks oxidation and phosphorylation by blocking the flow of protons through ATP synthase.
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Under aerobic conditions regeneration of cytosolic NAD+ from cytosolic NADH is accomplished by transferring electrons across the mitochondrial membrane barrier to the electron transport chain where the electrons are transferred to oxygen.

There are two different shuttle mechanisms whereby this transfer of electrons across the membrane to regenerate cytosolic NAD+ can be accomplished, the glycerol 3-phosphate shuttle and the malate-aspartate shuttle.

1) The glycerol 3-phosphate shuttle (Figure-1) functions primarily in skeletal muscle and brain. The shuttle takes advantage of the fact that the enzyme glycerol-3-phosphate dehydrogenase exists in two forms, a cytosolic form that uses NAD+ as cofactor and a mitochondrial FAD-linked form.

 

 Figure-1- showing glycerol-3-Phosphate shuttle. G3P- glycerol-3-P, DHAP- Dihydroxy acetone-Phosphate

Cytosolic glycerol-3-phosphate dehydrogenase uses electrons from cytosolic NADH to reduce the glycolytic intermediate dihydroxyacetone phosphate to glycerol 3-phosphate, thereby regenerating cytosolic NAD+. The newly formed glycerol 3-phosphate is released from the cytosolic form of the enzyme and crosses to and is bound to the mitochondrial FAD-linked glycerol-3-phosphate dehydrogenase, which is bound to the cytosolic side of the mitochondrial inner membrane. There the mitochondrial glycerol-3-phosphate dehydrogenase reoxidizes glycerol 3-phosphate to dihydroxyacetone phosphate (preserving mass balance) reducing its FAD cofactor to FADH2. Electrons are then passed  through complex II to coenzyme Q of the electron transport chain and on to oxygen generating two ATP molecules per electron pair and therefore per glycerol -3-phosphate.

2) The malate-aspartate shuttle, however, functions primarily in the heart, liver, and kidney (Figure 2). This shuttle requires cytosolic and mitochondrial forms of malate dehydrogenase and glutamate-oxaloacetate transaminase and two antiporters, the malate-α-ketoglutarate antiporter and the glutamate aspartate antiporter, which are both localized in the mitochondrial inner membrane. In this shuttle cytosolic NADH is oxidized to regenerate cytosolic NADby reducing oxaloacetate to malate by cytosolic malate dehydrogenase.(1)

 

Figure-2-Showing Malate Aspartate shuttle

Malate is transported into the mitochondrial matrix while α-ketoglutarate is transported out by the malate-α-ketoglutarate antiporter, a seeming mass unbalance(2).

Next malate is oxidized back to oxaloacetate producing NADH from NAD+ in the mitochondrial matrix by mitochondrial malate dehydrogenase (3).

Oxaloacetate cannot be transported per se across the mitochondrial membrane. It is, instead transaminated to aspartate from the NH3 donor glutamate by mitochondrial glutamate-oxaloacetate transaminase (4). Aspartate is transported out of the matrix whereas glutamate is transported in by the glutamate-aspartate antiporter (5)in the mitochondrial membrane, obviating the apparent mass unbalance noted above.

The last step of the shuttle is catalyzed by cytosolic glutamate-oxaloacetate transaminase regenerating cytosolic oxaloacetate from aspartate and cytosolic glutamate from α-ketoglutarate(6) both of which were earlier transported in opposing directions by the malate- α- ketoglutarate antiporter. 

The net effect of this shuttle is to transport electrons from cytosolic NADH to mitochondrial NAD+. Therefore, those electrons can be presented by the newly formed NADH to electron transport system complex I thereby producing three ATPs by oxidative phosphorylation.

Note that depending on which shuttle is used (i.e., which tissue is catalyzing glycolysis) either two or three ATPs are produced by oxidative phosphorylation per triose phosphate going through the latter steps of glycolysis.

 

                                             ATP Formation in the Catabolism of Glucose 

 

Pathway

Reaction Catalyzed by

Method of ATP Formation

ATP per Mol of Glucose

Glycolysis

Glyceraldehyde 3-phosphate dehydrogenase

Respiratory chain oxidation of 2 NADH

6*

Phosphoglycerate kinase

Substrate level phosphorylation

2

Pyruvate kinase

Substrate level phosphorylation

2

 

 Total yield

10

Consumption of ATP for reactions of hexokinase and phosphofructokinase

–2

 

 

Net 8

Citric acid cycle

Pyruvate dehydrogenase

Respiratory chain oxidation of 2 NADH

6

Isocitrate dehydrogenase

Respiratory chain oxidation of 2 NADH

6

α-Ketoglutarate dehydrogenase

Respiratory chain oxidation of 2 NADH

6

Succinate thiokinase

Substrate level phosphorylation

2

Succinate dehydrogenase

Respiratory chain oxidation of 2 FADH2

4

Malate dehydrogenase

Respiratory chain oxidation of 2 NADH

6

 

 

Net 30

 

Total per mol of glucose under aerobic conditions

38

 

Total per mol of glucose under anaerobic conditions

2

 

This assumes that NADH formed in glycolysis is transported into mitochondria by the malate shuttle.

If the Glycerol-phosphate shuttle is used, then only 2 ATP will be formed per mol of NADH. At the step of glyceraldehyde-3-P dehydrogenase-4 ATP will be produced. Hence the total will be-6+30= 36 ATP. ( 6 from Glycolysis and 30 from PDH complex and TCA cycle. Since this shuttle operates. in skeletal muscle and brain, hence the total yield per glucose mol will be 2 ATP less as compared to other tissues.

Clinical Significance

In nonaerobic glycolysis, as in the case when a tissue is subjected to an ischemic episode (i.e., myocardial infarction), neither the ATP produced by the shuttle nor the ATPs produced by normal passage of electrons through the electron transport chain are produced because of oxygen insufficiency.

Therefore glycolysis must increase in rate to meet the energy demand. In damaged tissue this increased rate is compromised. Moreover the shuttle mechanisms to regenerate NADfrom NADH formed by glycolysis are unavailable.

Glycolysis under ischemic conditions satisfies the requirement for NAD+ by reducing pyruvate, the glycolytic end product under normal conditions, to lactate with the reducing equivalents of NADH.

The new end product lactate accumulates in muscle cells under ischemic conditions and damages cell walls with its low pH causing rupture and loss of cell contents such as myoglobin and troponin I. These compounds as well as other end products combine to cause increased cell rupture and pain.

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Introduction

Most of the energy liberated during the oxidation of carbohydrates, fatty acids, and amino acids is made available within mitochondria as reducing equivalents (—H or electrons) .The NADH and FADH2 formed in glycolysis, fatty acid oxidation,and the citric acid cycle are energy-rich molecules because each contains a pair of electrons having a high transfer potential. The enzymes of the citric acid cycle and β-oxidation are contained in mitochondria, together with the respiratory chain, which collects and transports reducing equivalents, directing them to their final reaction with oxygen to form water. Hence Electron transport chain is operative only in the presence of oxygen, it is strictly aerobic.

Oxidative phosphorylation

The reduction of molecular oxygen to water yields a large amount of free energy that can be used to generate ATP. Oxidative phosphorylation is the process in which ATP is formed as a result of the transfer of electrons from NADH or FADH2 to O2 by a series of electron carriers. This process, which takes place in mitochondria, is the major source of ATP in aerobic organisms.

Components of Electron Transport Chain

Electrons flow through the respiratory chain through a redox span of 1.1V from NAD+/NADH to O2/2H2O, passing through three large protein complexes; NADH-Q oxidoreductase (Complex I), where electrons are transferred from NADH to coenzyme Q (Q) (also called Ubiquinone);Q-cytochrome c oxidoreductase (Complex III), which passes the electrons on to cytochrome c; and cytochrome c oxidase(Complex IV), which completes the chain, passing the electrons to O2and causing it to be reduced to H2O (Figure-1). Some substrates with more positive redox potentials than NAD+/NADH (eg, succinate) pass electrons to Q via a  fourth complex, succinate Q reductase (Complex II), rather than Complex I. The four complexes are embedded in the inner mitochondrial membrane, but Q and cytochrome c are mobile. Q diffuses rapidly within the membrane, while cytochrome c is a soluble protein.

Flavoproteins

Flavoproteins (are important components of Complexes I and II. The oxidized flavin nucleotide (FMN or FAD) can be reduced in reactions involving the transfer of two electrons (to form FMNH2 or FADH2),but they can also accept one electron to form the semiquinone. Iron-sulfur proteins (non-heme iron proteins, Fe-S) are found in Complexes I, II, and III. These may contain one, two, or four Fe atoms linked to inorganic sulfur atoms and/or via cysteine-SH groups to the protein. The Fe-S takes part in single electron transfer reactions in which one Fe atom undergoes oxidoreduction between Fe2+ and Fe3+.

Electron flow in ETC

NADH-Q oxidoreductase or Complex I is a large L-shaped multi-subunit protein that catalyzes electron transfer from NADH to Q, coupled with the transfer of  four H+across the membrane: Electrons are transferred from NADH to FMN initially, then to a series of Fe-S centers, and finally to Q (Figure-1). In Complex II(succinate -Q reductase), FADH2 is formed during the conversion of succinate to fumarate in the citric acid cycle and electrons are then passed via several Fe-S centres to Q.

Coenzyme Q or Ubiquinone (ubi)

Ubi accepts electrons and transports them to Complex III. Ubi is capable of moving in the membrane between Complex I and III, picking up electrons at Complex I and dropping them off at Complex III. As is the case with Complex I, Complex III contains a series of proteins that transport electrons. As the electrons are moved from carrier to carrier within Complex III, the energy that is released moves hydrogen ions out of the mitochondrion. Thus, Complex III also serves as a hydrogen ion pump. Once again, there must be a mechanism by which electrons are removed from Complex III. This done by a small peripheral membrane protein known as cytochrome c. Cytochrome c transports the electrons from Complex III to Complex IV.

 

Figure-1- Showing the components of electron transport chain

Reduced cytochrome c is oxidized by Complex IV (cytochrome c oxidase), with the concomitant reduction of O2 to two molecules of water:

This transfer of four electrons from cytochrome c to O2 involves two heme groups, a and a3,and Cu. Electrons are passed initially to a Cu centre (CuA), which contains 2 Cu atoms linked to two protein cysteine-SH groups (resembling an Fe-S), then in sequence to heme a, heme a3, a second Cu centre, CuB, which is linked to heme a3, and finally to O2.

By the time the electrons reach the final electron acceptor in Complex IV, they have very little energy left and need to be removed entirely from the system(otherwise the entire system will back up and be halted). Complex IV finally gives its electrons to molecular oxygen (O2), the final or terminal electron acceptor. The reduction of oxygen (remember, accepting electrons is a reduction reaction) results in the formation of a water molecule from the oxygen. For every pair of electrons donated to the ETC by an NADH, ½ O2(or one O atom) is reduced to form one water molecule. Two NADH’s would result in the use of 1 (½ + ½) O2 molecule, and so on. If there was no oxygen to take the electrons from Complex IV, the electrons would accumulate up the electron transport chain very quickly and NADH would no longer be able to donate electrons to the ETC (because the ETC would be filled with electrons).This would cause an accumulation of NADH and a shortage of NAD+(remember, NAD+ is regenerated when NADH donates its electrons to Complex I). Without NAD+, there would be no electron acceptors for the Krebs Cycle and all reactions that require NAD+ would stop. This would cause the entire Krebs Cycle to stop. Thus the Krebs Cycle would not run if there were no oxygen present, thus it is considered aerobic!

Proton Motive Force 

The flow of electrons from NADH or FADH2 to O2 through protein complexes located in the mitochondrial inner membrane leads to the pumping of protons out of the mitochondrial matrix.(Figure1) The resulting uneven distribution of protons generates a pH gradient and a transmembrane electrical potential that creates a proton-motive force (Figure -2). ATP is synthesized when protons flow back to the mitochondrial matrix through an enzyme complex (Figure -1). Thus, the oxidation of fuels and the phosphorylation of ADP are coupled by a proton gradient across the inner mitochondrial membrane.

 

Figure-2-showing the electrochemical gradient created by pumping of protons across the inner mitochondrial membrane

In other words, the electron-motive force is converted into a proton-motive force and, finally, the proton-motive force is converted into phosphoryl transfer potential.

Like NADH, FADH2 contains electrons (and energy) from oxidation reactions. Remember that FADH2, however, contains less energy than does NADH. Because of this, FADH2 is unable to donate its electrons to Complex I of the ETC. Instead, FADH2 donates its electrons to Complex II.Complex II eventually passes the electrons on to Complex III via Ubiquinone.From then on, the electrons follow the same pathway as do those from NADH.Complex II, it turns out, does not move protons as the electrons are transported. This means that every NADH that donates electrons to the ETC will cause more hydrogen ions to be transferred than does each FADH2.

P: O ratio

There is a net direct capture of two high-energy phosphate groups in the glycolytic reactions. Two more high-energy phosphates per mole of glucose are captured in the citric acid cycle during the conversion of succinyl CoA to succinate. All of these phosphorylation occur at the substrate level. When substrates are oxidized via Complexes I, III, and IV in the respiratory chain (ie, via NADH), 3 mol of ATP are formed per half mol of O2consumed; ie, the P:O ratio = 3. On the other hand, when a substrate (e.g.succinate) via FADH2 is oxidized through Complexes II, III, and IV, only 2 mol of ATP are formed; ie, P:O =2. These reactions are known as oxidative phosphorylation at the respiratory chain level. Taking these values into account, it can be estimated that nearly 90% of the high energy phosphates produced from the complete oxidation of 1 mol glucose is obtained via oxidative phosphorylation coupled to the respiratory chain

Inhibition of Oxidative phosphorylation

Oxidative phosphorylation is susceptible to inhibition at all stages of the process.  

1) Site specific inhibitors -Specific inhibitors of electron transport for example, rotenone and amobarbital block electron transfer in NADH-Q oxidoreductase and thereby prevent the utilization of NADH as a substrate. In contrast, electron flow resulting from the oxidation of succinate is unimpaired, because these electrons enter through QH2, beyond the block. BAL(British Anti Lewisite), Antimycin A  interfere with electron flow from cytochrome b in Q-cytochrome c oxidoreductase. Furthermore,electron flow in cytochrome c oxidase can be blocked by cyanide (CN-),azide (N3 -), and carbon monoxide (CO). Cyanide and azide react with the ferric form of heme a 3, whereas carbon monoxide inhibits the ferrous form. Inhibition of the electron-transport chain also inhibits ATPsynthesis because the proton-motive force can no longer be generated. Malonate is a competitive inhibitor of Complex II

2) ATP synthase- also can be inhibited.Oligomycin and dicyclohexyl carbodiimide(DCCD) prevent the influx of protons through ATP synthase. If actively respiring mitochondria are exposed to an inhibitor of ATP synthase, the electron transport chain ceases to operate. Indeed, this observation clearly illustrates that electron transport and ATP synthesis are normally tightly coupled.

3)Inhibition of ATP-ADP translocase-ATP-ADP translocase is specifically inhibited by very low concentrations of Atractyloside(a plant glycoside) or bongregate (an antibiotic from a mold).Unavailability of ADP also inhibits the process of ATP formation. This is because oxidation and phosphorylation are tightly coupled; ie, oxidation cannot proceed via the respiratory chain without concomitant phosphorylation of ADP.

4)Uncouplers of Oxidative phosphorylation- This tight coupling of electron transport and phosphorylation in mitochondria can be disrupted(uncoupled) by 2,4- dinitrophenol ,2,4  dinitrocresol  and certain other acidic aromatic compounds.These substances carry protons across the inner mitochondrial membrane. In the presence of these uncouplers, electron transport from NADH to O2 proceeds in a normal fashion, but ATP is not formed by mitochondrial ATP synthase because the proton-motive force across the inner mitochondrial membrane is dissipated. This loss of respiratory control leads to increased oxygen consumption and oxidation of NADH. Indeed, in the accidental ingestion of uncouplers, large amounts of metabolic fuels are consumed, but no energy is stored as ATP. Rather, energy is released as  heat.

The regulated uncoupling of oxidative phosphorylation is a biologically useful means of generating heat. The uncoupling of oxidative phosphorylation is a means of generating heat to maintain body temperature in hibernating animals,in some newborn animals (including human beings), and in mammals adapted to cold. Brown adipose tissue, which is very rich in mitochondria (often referred to as brown fat mitochondria), is specialized for this process of non shivering thermogenesis. The inner mitochondrial membrane of these mitochondria contains a large amount of uncoupling protein (UCP), here UCP-1, or thermogenin,a dimer of 33-kd subunits that resembles ATP-ADP translocase. UCP-1 forms a pathway for the flow of protons from the cytosol to the matrix. In essence, UCP-1 generates heat by short-circuiting the mitochondrial proton battery.

Thus,the manner in which biologic oxidative processes allow the free energy resulting from the oxidation of foodstuffs to become available and to be captured is stepwise, efficient, and controlled—rather than explosive,inefficient, and uncontrolled, as in many non biologic processes. The remaining free energy that is not captured as high-energy phosphate is liberated as heat.This need not be considered “wasted,” since it ensures that the respiratory system as a whole is sufficiently exergonic, allowing continuous unidirectional flow and constant provision of ATP. It also contributes to maintenance of body temperature.

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