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Biological Oxidation

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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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As electrons are received and passed down the transport chain, the electron carriers are first reduced with the acceptance of the electron and then oxidized with loss of the electron. A patient poisoned by which of the following compounds has the most highly reduced state of most of the respiratory chain carriers?

A. Antimycin A

B. Rotenone

C. Carbon monoxide

D. Puromycin

E. Chloramphenicol

The right answer is (c) – Carbon monoxide.

Basic concept

The electron transport chain contains three proton pumps linked by two mobile electron carriers. At each of these three sites (NADH-Q reductase, cytochrome c reductase and cytochrome oxidase); the transfer of electrons down the chain powers the pumping of the protons across the inner mitochondrial membrane (figure). As electrons pass through complexes I, III, and IV, protons are pumped across the inner membrane from the matrix to the intermembrane space. This sets up an electrochemical gradient consisting of a proton gradient (chemical), and a membrane potential (because the proton carries a positive charge). The electrochemical gradient represents a form of stored energy derived from the oxidation of NADH (or succinate). A physically distinct complex (ATP synthase or complex V) at a separate location in the inner membrane can exploit the electrochemical gradient to carry out the endergonic ATP synthesis (by oxidative phosphorylation).

 Inhibition of electron transport chain

The blockage of electron transfers by specific point inhibitors leads to a buildup of highly reduced carriers behind the block because of the inability to transfer electrons across the block.

Antimycin A blocks the complex III, Rotenone blocks complex I and Carbon monoxide (As well as cyanide, azide, hydrogen sulfide) block complex IV. Therefore a carbon monoxide inhibition leads to a highly reduced state of all the carriers of the chain. Puromycin and Chloramphenicol are inhibitors of protein synthesis and have no direct effect upon the electron transport chain.

 ETC

Figure-1- Flow of electrons in the electron transport chain and the oxidative phosphorylation

 

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An unskilled worker in a water garden was sent to sweep up a spill of a white powder in the storage shed. Later he was found with labored breathing and convulsions. On further examination, the white powder was identified as rotenone. Respiratory distress is induced on rotenone exposure because it inhibits the complex that catalyzes which of the following?

A. Electron transfer from NADH to coenzyme Q

B. Oxidation of coenzyme Q

C. Reduction of cytochrome c

D. Electron transfer from cytochrome c to cytochrome a1/a3

E. Electron transfer from cytochrome a1/a3 to oxygen

 

The right answer is A) – Electron transfer from NADH to coenzyme Q

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 four large protein complexes;

i) NADH-Q oxidoreductase (Complex I), where electrons are transferred from NADH to coenzyme Q (also called Ubiquinone);

ii) Succinate Q reductase (Complex II)-Some substrates with more positive redox potentials than NAD+/NADH (e.g., succinate) pass electrons to Q via succinate Q reductase (Complex II), rather than Complex I.

iii) Q-cytochrome c oxidoreductase (Complex III), which passes the electrons on to cytochrome c; and

iv) Cytochrome c oxidase (Complex IV), which completes the chain, passing the electrons to O2 and causing it to be reduced to H2O (Figure-1).

Mobile complexes

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.

 ETC

Figure-1- Components of electron transport chain. The three complexes I, III and IV acts as proton pumps, ATP is synthesized when protons flow back to the mitochondrial matrix through ATP synthase complex.

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 (figure-2)

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-2). 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. The electrons are removed from Complex III by a small peripheral membrane protein known as cytochrome c. Cytochrome c transports the electrons from Complex III to Complex IV.

Reduced cytochrome c is oxidized by Complex IV (cytochrome c oxidase).

This transfer of four electrons from cytochrome c to O2 involves two heme groups, a and a3, and Cu. 

Complex IV finally gives its electrons to molecular oxygen (O2), the final or terminal electron acceptor. The reduction of oxygen results in the formation of a water molecule from the oxygen.

 

 Flow of electrons

Figure-2- Flow of electrons in the electron transport chain

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. The central portion of the rotenone structure resembles the isoalloxazine ring of the FMN molecule, and when it binds to complex I, rotenone prevents the transfer of electrons from NADH to coenzyme Q. In contrast, electron flow resulting from the oxidation of succinate is unimpaired, because these electrons enter through QH2, beyond the block. Malonate is a competitive inhibitor of Complex II

BAL (British Anti Lewisite), Antimycin A interferes 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 ATP synthesis because the proton-motive force can no longer be generated.

 

 

 

 

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An -8 year-old boy is seen by an ophthalmologist for difficulties in seeing in all visual fields as well as slow eye movements. The ophthalmologist finds pigmentary retinopathy and ophthalmoplegia. The child is suspected to have Kearns- Sayre syndrome, a disorder due to a mutation in complex II of ETC. The electron transport from which substance would be impaired?

A. Malate

B. Isocitrate

C. Succinate

D. Pyruvate

E. Alpha Keto glutarate

Answer- The correct answer is C- Succinate.

Electrons flow through the respiratory chain through a redox span of 1.1 V from NAD+/NADH to O2/2H2O passing through three large protein complexes;

1) NADH-Q oxidoreductase (Complex I), where electrons are transferred from NADH to coenzyme Q (Q) (also called ubiquinone)

2) Q-cytochrome c oxidoreductase (Complex III), which passes the electrons on to cytochrome c; and

3) Cytochrome c oxidase (Complex IV), which completes the chain, passing the electrons to O2 and causing it to be reduced to H2O .

Some substrates with more positive redox potentials than NAD+/NADH (e.g., succinate) pass electrons to Q via, 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.

ETC

Figure- Flow of electrons through the respiratory chain complexes.

Malate, Isocitrate and alpha ketoglutarate are intermediates of TCA cycle, the electrons flow from them to NAD+ forming NADH that is regenerated back to its oxidized form by passing electrons to complex I of ETC. Similarly electron flow from Pyruvate is also through complex I.

Electron from Succinate pass to FAD forming FADH2 that is regenerated back to its oxidized form after passing its electrons to complex II of ETC.

Thus the correct answer for the given problem is Succinate.

 

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Q.1- If the mitochondria were blocked at the site of NADH oxidation and were treated with succinate as substrate, what would the P: O ratio is?

A. Zero

B. One less than normally produced by succinate

C. Same as that normally produced by succinate

D. One more than normally produced by succinate

E. Higher than normal because of the excessive heat produced from uncoupling

Q.2- If the oxidative phosphorylation was uncoupled in the mitochondria, what would one expect?

A. A decreased concentration of ADP in the mitochondria

B. Increased inorganic phosphate in the mitochondria

C. A decreased oxidative rate

D. A decreased production of heat

E. Increased transport of ADP from the cytosol to the mitochondrial matrix

Q.3- If the rotenone is added to the mitochondrial electron transport chain:

A. P: O ratio of NADH is reduced from 3:1 to 2:1

B. Rate of NADH oxidation is diminished to two- thirds of its initial value

C. Succinate oxidation remains normal

D. Oxidative phosphorylation is uncoupled at site I

E. Electron flow is inhibited at site II

Q.4- If 2, 4 dinitro phenol is added to tightly coupled mitochondria that are actively oxidizing succinate:

A. Electron flow will continue but ATP synthesis will not occur

B. Electron flow will continue but ATP synthesis will be increased

C. Electron flow will cease, but ATP synthesis will continue

D. Both Electron flow and ATP synthesis will be ceased

E. Subsequent addition of Oligomycin will cause ATP hydrolysis

Q.5- The prosthetic group of NADH dehydrogenase:

A. FMN

B. NADH

C. FAD

D. NADPH

E. Iron

Q.6- In substrate level phosphorylation

A. The substrate reacts to form a product containing a high energy bond

B. ATP synthesis is linked to dissipation of proton gradient

C. High energy intermediate compounds cannot be isolated

D. Oxidation of one molecule of substrate is linked to synthesis of more than one ATP molecule

E. Only mitochondrial reactions participate in ATP formation

Q.7- The chemiosmotic hypothesis involves all of the following except

A. A membrane impermeable to protons

B. Electron transport by the respiratory chain pumps protons out of the mitochondria

C. Proton flow in to the mitochondria depends on the presence of ADP and Pi

D. ATPase activity is reversible

E. Only proton transport is strictly regulated, other positively charged ions can diffuse freely across the mitochondrial membrane

Q.8- The effect of Valinomycin on oxidative phosphorylation involves all of the following except-

A. The net yield of ATP decreases

B. Rate of oxygen consumption increases

C.  Excessive heat is released

D. pH gradient across the inner mitochondrial membrane decreases

E. The rate of flow of electrons increases

Q.9- Which of the following ETC components accept only one electron?

A. Coenzyme Q

B. Cytochrome b

C. FAD

D.FMN

E. O2

Q.10- Which of the following has highest redox potential in the respiratory chain?

A. O2

B. Ubiquinone

C. NAD

D.FMN

E. FAD

Key to answers

1)-C, 2)-B, 3)-C, 4)-A, 5)-A, 6)-A, 7)-E, 8)-D, 9) – B, 10)-A.

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Q.1- All of the following except one are NAD+ requiring enzymes –

A. Acyl co A dehydrogenase

B. Glyceraldehyde-3-P dehydrogenase

C. Pyruvate dehydrogenase complex

D. Malate dehydrogenase

E. Lactate dehydrogenase

Q.2- Which one of the following enzymes catalyzes substrate level phosphorylation in TCA cycle

A. Malate dehydrogenase

B. Succinate Thiokinase

C. Succinate dehydrogenase

D. Alpha keto dehydrogenase complex

E. Isocitrate dehydrogenase

Q.3- The major metabolic consequence of perturbation of the electron transfer in mitochondria is which of the following?

A. Increased production of NADPH

B. Increased oxidation of NADH

C. Increased reduction of O2 to H2O

D. Decreased regeneration of NAD+

E. Decreased reduction of FAD

Q.4- An unskilled worker in a water garden/plant nursery was sent to sweep up a spill of a white powder in the storage shed. Later he was found with labored breathing and convulsions. On further examination, the white powder was identified as rotenone. Respiratory distress is induced on rotenone exposure because it inhibits the complex that catalyzes which of the following?

A. Electron transfer from NADH to coenzyme Q

B. Oxidation of coenzyme Q

C. Reduction of cytochrome c

D. Electron transfer from cytochrome c to cytochrome a1/a3

E. Electron transfer from cytochrome a1/a3 to oxygen

Q.5- Which of the following procedures best describes the emergency intervention for cyanide poisoning?

A. Decrease the partial pressure of oxygen

B. Treatment with nitrites to convert hemoglobin to methemoglobin.

C. Treatment with thiosulfate to form thiocyanate

D. Use of N-acetyl cysteine taken orally

E. Use of Antioxidants

Q.6- Inhibition of oxidative phosphorylation by cyanide ion leads to increases in which of the following?

A. Gluconeogenesis to provide more glucose for metabolism

B. Transport of ADP into the mitochondria

C. Utilization of fatty acids substrates to augment glucose utilization

D. Utilization of ketone bodies for energy generation

E. Lactic acid in the blood causing acidosis

Q.7- 27-year-old male with acute appendicitis undergoing a halothane inhaled anesthetic with acute onset of hyperthermia, tachypnea, respiratory acidosis, hyperkalemia, and family history of similar events. The tentative diagnosis is malignant hyperthermia (MH). Which of the following processes gets affected by Halothane?

A. Inhibition of NADH-Q oxidoreductase (Complex I)

B. Inhibition of Q-cytochrome c oxidoreductase (Complex III)

C. Inhibition of succinate Q reductase (Complex II)

D. Inhibition of ADP/ATP transporter

E. Uncoupling of oxidative phosphorylation

Q.8- The rate of respiration of mitochondria can be controlled by the availability of:

A. ADP

B. ATP

C. FMN

D.FAD

E. NAD +

Q.9- Atractyloside inhibits oxidative phosphorylation by inhibiting:

A. NADH-Q oxidoreductase (Complex I)

B. Q-cytochrome c oxidoreductase (Complex III)

C. Succinate Q reductase (Complex II)

D. ADP/ATP transporter

E. Cytochrome a1-a3 oxidase

Q.10- The enzyme that catalyzes the direct transfer and incorporation of oxygen into a substrate molecule is known as:

A. Oxidase

B. Oxygenase

C. Peroxidase

D. Reductase

E. Hydratase

Key to Answers

1)-A, 2) -B, 3) – D, 4)-A, 5)-B, 6)-E, 7)-E, 8)-A, 9)-D, 10)-B

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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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An 18-year-old college student is brought to the emergency room unconscious, with a very high serum alcohol level. Alcohol metabolism can result in high NADH levels. When NADH enters the electron transport chain, which of the following is the correct order in which electron transfer occurs ?

a) NADH, coenzyme Q, cytochrome c, FMN,O2

b) NADH, cytochrome c, coenzyme Q, FMN,O2

c) NADH, FMN, coenzyme Q, cytochrome c, O2

d) NADH, cytochrome c, FMN, cytochrome c, O2

e) NADH, FMN, cytochrome c, coenzyme Q, O2

 

The right answer is (c).The electron flow is from NADH to O2 passing through FMN, coenzyme Q (ubiquinone) and cytochrome c (Figure-1). See the details below-

 

Overview of ETC

 Figure-1  – An overview of Electron transport chain . FeS represents iron sulfur center, and cytochrome b,c1 are components of complex III.

Basic concept of electron transport chain

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 (electron transport chain), which collects and transports reducing equivalents, directing them to their final reaction with oxygen to form water (Figure-2)

Role of ETC in ATP formation

 Figure-2- The energy trapped in the food is passed on to electron transport chain after a series of oxidative processes, in the form of reducing equivalents, where finally energy is released as ATP

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 (Figure-3)

1) NADH-Q oxidoreductase (Complex I), where electrons are transferred from NADH to coenzyme Q (Q) (also called Ubiquinone);

2) Q-cytochrome c oxidoreductase (Complex III), which passes the electrons on to cytochrome c; and

3) Cytochrome c oxidase (Complex IV) ,which completes the chain, passing the electrons to Oand causing it to be reduced to H2O (Figure-1).

4) Succinate Q reductase (Complex II),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.

 

Components of ETC

 Figure-3- Showing the components of electron transport chain. Arrows show the flow of protons.

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-4).

NADH dehydrogenase complex

 Figure-4NADH-Q oxidoreductase 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

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 (Figure-3 and 5).

Flow of electrons in ETC

Figure-5- Showing the flow of electrons through four complexes. Coenzyme Q and cytochrome c are mobile carriers. Complex V is ATP synthase complex, meant for ATP production.

Iron-sulfur proteins (non-heme iron proteins, Fe-S) are found in Complexes I, II, and III (Figure-5).These may contain one, two, or four Fe atoms linked to inorganic sulfur atoms and/or via cysteine-SH groups to the protein (Figure-6). The Fe-S takes part in single electron transfer reactions in which one Fe atom undergoes oxidoreduction between Fe2+ and Fe3+.

 

Iron -sulphur centers

Figure-6- Iron sulfur proteins (Fe-S). (A) The simplest Fe-S with one Fe bound by four cysteines. (B) 2Fe-2S center. (C) 4Fe-4S center.

Flavoproteins (are important components of Complexes I and II (Figure-4). 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.

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 (Figure-3 and 5).

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 (Figure-3).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.

Reduced cytochrome c is oxidized by Complex IV (cytochrome c oxidase), with the concomitant reduction of O2 to two molecules of water(Figure-3 and 4).

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 . Complex IV finally gives its electrons to molecular oxygen (O2), the final or terminal electron acceptor. The reduction of oxygen results in the formation of a water molecule from the oxygen (Figure-3 and 5).

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.(Figure-3) .The resulting uneven distribution of protons generates a pH gradient and a transmembrane electrical potential that creates a proton-motive force (Figure -7). ATP is synthesized when protons flow back to the mitochondrial matrix through an enzyme complex (Figure -3 and 4). Thus, the oxidation of fuels and the phosphorylation of ADP are coupled by a proton gradient across the inner mitochondrial membrane.

Proton motive force

 Figure-7-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. 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.

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.

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 (i.e., via NADH), 3 mol of ATP are formed per half mol of O2 consumed; 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; i.e., 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.

To be continued……

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Q.1- Choose a site along the electron transport chain out of the following that is not coupled to ATP synthesis-

a) NADH- coenzyme Q (CoQ) reductase

b) Succinate-Co Q reductase

c) Cytochrome bc1 reductase

d) Cytochrome oxidase

e) None of the above.

Q.2- Choose a statement out of the following that best describes the uncouplers of oxidative phosphorylation-

a) Uncouple ATP synthesis with phosphoenol pyruvate

b) Uncouple ATP/ADP translocation

c) Uncouple electron transport from oxygen reduction

d) Uncouple electron transport with ATP synthesis

e) Uncouple ATP synthesis with phosphoglycerate

Q.3- Which of the following statements best describes the mechanism of action of Oligomycin ?

a)It inhibits NADH dehydrogenase

b) It is an uncoupler of oxidative phosphorylation

c) It inhibits ATP/ADP transporter

d) It is an inhibitor of cytochrome oxidase

e) It blocks the flow of protons through ATP synthase complex

Q.4- ADP transport in to the mitochondrial matrix–

a) is an active transport process

b) is directly inhibited by valinomycin

c) is carried out through a shuttle present in outer mitochondrial membrane

d) is accompanied by ATP export from the mitochondrial matrix

e) is inhibited by Rotenone

Q.5- Which of the following enzymes catalyzes the conversion of Hydrogen peroxide to water ?

a) Super oxide dismutase

b) Cytochrome P 450

c) Catalase

d) Hydratase

e) Dioxygenase

Q.6- Which of the following ETC components accepts only one electron ?

a) Oxygen

b) FMN

c) FAD

d) Cytochrome b

e) Coenzyme Q

Q.7- Which of the following is a component of Succinate dehydrogenase in Electron transport chain ?

a) Niacin

b) FMN

c) FAD

d) Coenzyme Q

e) Lipoic acid

Q.8- MELAS is a mitochondrial disorder characterized by mitochondrial encephalopathy , lactic acidosis and stroke-like episode. It is an inherited condition due to NADH:Q oxidoreductase (Complex I) or cytochrome oxidase (Complex IV) deficiency, caused by a mutation in mitochondrial DNA and may be involved in Alzheimer’s disease and diabetes mellitus. Due to lack of functional mitochondria, what would be the net ATP that would be produced from one molecule of Glucose ?

a) 1

b) 2

c) 4

d) 8

e) 0

Q.9- An 18- year -old cricket player sustains a compound fracture on the field. He is taken for surgery, during which anesthesiologist notes a significantly increased body temperature (104OF). There is suspicion of malignant hyperthermia. Which of the following components of ETC is likely to be responsible for this phenomenon ?

a) Complex I

b) Complex II

c) Complex III

d) Complex IV

e) ATP synthase complex

Q.10- A 43- year-old woman diagnosed with early stage breast cancer elects to have a lumpectomy followed by radiation therapy because this regimen has been shown to be equivalent to mastectomy in such patients. Radiations work in part by-

a) Inhibiting NADH – cytochrome C reductase

b) Inhibiting cytochrome b-c1 complex

c) Generating reactive oxygen species

d) Oxidizing glutathione

e) Inhibiting ATP synthase complex

Q.11- An -8 year-old boy is seen by an ophthalmologist for difficulties in seeing in all visual fields as well as slow eye movements. The ophthalmologist finds pigmentary retinopathy and ophthalmoplegia. The child is suspected to have Kearns- Sayre syndrome, a disorder due to a mutation in complex II of ETC. The electron transport from which substance would be impaired ?

a) Malate

b) Isocitrate

c) Succinate

d) Pyruvate

e) Alpha keto glutarate

Q.12- A 63- year-old man with a strong family history of Parkinson’s disease begins to show the signs of the disease “pin rolling tremors”. He visits his neurologist, who guides him about the recent research about coenzyme Q, that may stall the development of the disease. This component of ETC normally –

a) receives electrons directly from NADH

b) receives electrons directly from complex IV

c) receives electrons directly from FMN

d) transports ATP to the cytoplasm

e) Contains heme

 

Key to answers-

1)- b, 2)- d, 3)- e, 4)- d, 5)- c, 6)- d, 7)- c, 8)- b, 9)-e, 10)-c , 11)-c , 12)- c

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1. Dehydrogenases catalyze the transfer of hydrogen from one substrate to another in a coupled oxidation-reduction reaction .These dehydrogenases are specific for their substrates but often utilize common coenzymes or hydrogen carriers, e.g. NAD+. Which out of the following is not a niacin ( NAD+) dependent dehydrogenase ?

a) Pyruvate dehydrogenase

b) Glycerol-3-P dehydrogenase( Mitochondrial )

c) Glyceraldehyde-3-P dehydrogenase

d) Lactate dehydrogenase

e) Alpha keto glutarate dehydrogenase

2. The cytochromes are iron-containing hemoproteins in which the iron atom oscillates between Fe3+ and Fe2+ during oxidation and reduction. Most of the cytochromes of the electron transport chain are classified as dehydrogenases . Which out of the following cytochromes is not a dehydrogenase ?

a) NADH-Q oxidoreductase (Complex I)

b) Q-cytochrome c oxidoreductase (Complex III)

c) cytochrome c oxidase (Complex IV)

d) Succinate Q reductase (Complex II)

e) cytochrome c

3. A 2-year – old child has been brought to pediatric OPD with the complaint of passage of black colored urine. A probable diagnosis of Alkaptonuria has been made. Alkaptonuria is a congenital disorder of deficiency of Homogentisic acid oxidase. Homogentisic acid oxidase is an enzyme of tyrosine metabolic pathway and functionally it is –

a) A dehydrogenase

b) An oxidase

c) Mono oxygenase

d) Di oxygenase

e) Hydroperoxidase

4. A 23 -year -old male has been brought to medical OPD with jaundice and passage of dark colored urine. History reveals that he took antibiotic sulphonamides for a sore throat and ever since he developed the symptoms. There is history of previous such episodes also . A probable diagnosis of Glucose-6-P dehydrogenase deficiency has been made. NADPH  produced from the activity of this enzyme is required for the reductive processes in the body. Reduction of oxidized glutathione is one of such processes. G-SH (Reduced glutathione) is required for the activity of a selenium containing peroxidase, an enzyme required for the maintenance of the integrity of the red blood cell membrane. The reduced activity of this enzyme is responsible for hemolytic anemia in such patients. Which of the following is not a product of this enzyme catalyzed reaction ?

a) Hydrogen peroxide

b) Water

c) Oxidized substrate

d) NADP+

e) Oxidized glutathione

5. A chronic alcoholic has been brought to medical emergency. Blood biochemistry reveals lactic acidosis. Deficiency of pyruvate dehydrogenase complex is suspected. Pyruvate dehydrogenase complex (PDH complex) is a multienzyme complex, that requires, thiamine, Pantothenic acid, lipoic acid, riboflavin and niacin for its action and catalyzes the oxidative decarboxylation of pyruvate to from acetyl co A. During this process of oxidation what is the expected P:O ratio ?

a) 3:1

b) 6:1

c) 4:1

d) 12 :1

e) 38 :1

6. For each acetyl Co-A oxidized by the citric acid cycle, what is the energy gain by  substrate level phosphorylation ?

a) 2 ATP

b) 4 ATP

c) 6 ATP

d) 1 ATP

e) Zero ATP

7.Which of the following enzymes catalyzes a reaction with the resultant  P:O ratio of 2:1 ?

a) Glycerol-3-P-dehydrogenase (Cytosolic)

b) Succinate dehydrogenase

c) Malate dehydrogenase

d) Lactate dehydrogenase

e) Glucose-6-P dehydrogenase

8. Which of the following compounds inhibits oxidative phosphorylation by inhibiting the transporter of ADP into and ATP out of the mitochondrion ?

a) Malonate

b) Oxamate

c) Atractyloside

d) Barbiturates

e) Cyanide

9. The energy of oxidation is initially trapped as a high-energy phosphate compound and then used to form ATP. Which of the following intermediates of glycolysis is a high energy compound ?

a) Fructose-6-P

b) Glyceraldehyde-3-P

c) Fructose-1,6 bisphosphate

d) Glucose-6-P

e) Phosphoenol pyruvate

10. ATP is a nucleotide consisting of an adenine, a ribose, and a triphosphate unit. The active form of ATP is usually a complex of ATP with Mg2+ or Mn2+ . ATP is an energy-rich molecule because its triphosphate unit contains two phosphoanhydride bonds. A large amount of free energy of the amount ——– approximately  is liberated when ATP is hydrolyzed to adenosine diphosphate (ADP) and orthophosphate (Pi) .

a) 12.3 kcal/mol

b) 10.9 kcal/mol

c) 6.8 kcal/mol

d) 7.3 kcal/mol

e) 8.3 kcal/mol

11.  Which of the components of electron transport chain does not contain Iron sulfur center?

a) NADH dehydrogenase complex

b) Cytochrome a-a3 oxidase

c) Succinate dehydrogenase

d) Cytochrome bc1-c reductase

12. Aspirin in a high dosage produces hyperthermia, despite the fact that it is itself an antipyretic agent. The reason for this effect is-

a) Aspirin affects hypothalamic functions

b) It uncouples oxidative phosphorylation

c) It stimulates metabolism

d) It is itself metabolized to high energy compound

e) All of the above.

13. Choose the incorrect statement  out of the following about complete oxidation of glucose in the presence of oxygen-

a) In RBC s nearly 90% of the energy is obtained via oxidative phosphorylation

b) 1,3 Bisphosphoglycerate is a high energy compound

c) The conversion of phosphoenol pyruvate to pyruvate  yields 2 ATP

d) Net energy output in skeletal muscle is 36 ATP

e) 10 % of the total energy is obtained by substrate level phosphorylation in most tissues

14. Choose the correct statement out of the following  about Iron Sulfur centers-

a) Iron-sulfur proteins (non-heme iron proteins, Fe-S)  are found in Complexes IV

b) These may contain five  iron atoms linked to inorganic sulfur atoms

c) Iron may be linked through  cysteine-SH groups to the protein

d) The Fe-S take part in two electron transfer reactions

e) The Fe atom always  remains in the reduced form ( Fe2+) .

Key to answers- 1)-b , 2)-c, 3)-  d, 4)- a, 5- b, 6)-a, 7)-b, 8)-c, 9)-e,10)-d, 11)-b, 12-b, 13)-a, 14)-c.

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Case Study

An elderly couple was brought by ambulance to the emergency department after their daughter noticed that they were both acting “strangely.” The couple had been in good health prior to the weekend. Their daughter had gone out to spend the week-end with her friends.

The couple had been snowed in at their house until the snowplows cleared the roads. They had plenty of food and were kept warm by a furnace and blankets. On reaching home after two days, their daughter noticed that they both were complaining of bad headaches, confusion, fatigue, and some nausea.

On arrival to the emergency department, both patients were afebrile with normal vital signs and O2 saturation of 99 percent on 2 L of O2 by nasal cannula. Their lips appeared to be very red. Both patients were slightly confused but otherwise oriented. The physical examinations were within normal limits. Carboxyhemoglobin levels were drawn and were elevated.

What is the most likely cause of these patients’ symptoms?

Case details

The elderly couple is most probably suffering from CO poisoning. The cause might be the accidental exposure to CO by the burning of the furnace in the closed room. The symptoms, of headache, confusion, fatigue, nausea and red lips are suggestive of CO poisoning. Since the level of carboxy hemoglobin is also high, so the diagnosis is confirmed that the couple is suffering from CO poisoning.

Carbon monoxide (CO) poisoning, one of the most common fatal poisonings, occurs by inhalation. Carbon monoxide is a colorless, odorless gas produced by the combustion of carbon-containing materials. Poisoning may occur as a result of suicidal or accidental exposure to automobile exhaust, smoke inhalation in a fire, or accidental exposure to an improperly vented gas heater or other appliance. Inhaling tobacco smoke results in CO in the blood but not enough to cause poisoning. 

Mechanisms of CO toxicity are not completely understood. They appear to involve the following

1)   Carbon monoxide avidly binds to hemoglobin, with an affinity approximately 250 times that of oxygen. This results in displacement of O2 from Hb resulting in reduced oxygen-carrying capacity and altered delivery of oxygen to cells.

2)   Binding of CO to hemoglobin causes an increased binding of oxygen molecules at the 3 other oxygen-binding sites, resulting in a leftward shift in the oxyhemoglobin dissociation curve and decreasing the availability of oxygen to the already hypoxic tissues.

3)   Inhibition of mitochondrial electron transport chain

4)   Possibly direct toxic effects on brain tissue

CO binds to cardiac myoglobin with an even greater affinity than to hemoglobin; the resulting myocardial depression and hypotension exacerbates the tissue hypoxia. Toxicity primarily results from cellular hypoxia caused by impedance of oxygen delivery.

The transfer of Oto enzymes requiring O2 is also inhibited. Several different sites within the body get affected but its most profound impact is on the organs (eg, brain, heart) with the highest oxygen requirement. Following severe intoxication, patients display central nervous system (CNS) pathology, including white matter demyelination.

 Inhibition of Electron Transport Chain

CO toxicity causes impaired oxygen delivery and utilization at the cellular level. CO disrupts the O2-dependent step of the electron transport chain, leading to unavailability of ATP. In the electron transport chain, only complex IV (cytochrome oxidase) interacts directly with O2. As with hemoglobin, CO has a higher affinity for cytochrome oxidase than O2. Thus CO binds tightly to cytochrome oxidase and inhibits the binding of O2  This results in inhibition of electron transport chain with the resultant inhibition of  phosphorylation of adenosine diphosphate (ADP), to form adenosine triphosphate (ATP). This becomes more profound as additional molecules of cytochrome oxidase are bound by CO. 

Clinical symptoms- Symptoms tend to correlate well with the patient’s peak blood carboxy hemoglobin levels. Many symptoms are nonspecific.

  • Headache and nausea can begin when levels are 10 to 20%.
  • Levels > 20% commonly cause vague dizziness, generalized weakness, difficulty concentrating, and impaired judgment.
  • Levels > 30% commonly cause dyspnea during exertion, chest pain (in patients with coronary artery disease), and confusion.
  • Higher levels can cause syncope and  seizures,
  • Hypotension, coma, respiratory failure, and death may occur, usually when levels are > 60%.

Management- The binding of CO to hemoglobin is fully dissociable, and dissociation requires ventilation. Patients should be removed from the source of CO and stabilized as necessary. They are given 100% Oand treated supportively.

 Utilization of 100 percent O2 accelerates the washout of CO. Use of hyperbaric chambers with pressures up to 2 atmospheres speeds up the CO washout process even more. Addition of 5 to 7 percent CO2 to the O2 is sometimes used as a prompt to ventilatory exchange.

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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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Dear students,
Here are the links provided to you for your better understanding of oxidative phosphorylation

http://www.wiley.com/college/pratt/0471393878/student/animations/oxidative_phosphorylation/index.html

http://www.learnerstv.com/animation/animation.php?ani=177&cat=biology

(If clicking on the link doesn’t work, try copying and pasting it into your browser).

These are not my creations, please note.

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Q.1-Out of the following respiratory chain components indicate which  one is a mobile carriers of electrons?

a) Cytochrome oxidase

b) NADH-Q reductase

c) Ubiquinone

d) Succinate dehydrogenase                                             

Q.2-Choose the incorrect statement about redox potential

a) The redox potential of a system (E0) is usually compared with the potential of the hydrogen electrode

b) The components of electron transport chain are organized in terms of their redox potential.

c) NADH/NAD+ redox pair has the least redox potential of -0.42 volts

d) Oxygen/H2O redox pair has the highest redox potential of +.82 volts

Q.3-All are flavoproteins except one, choose the odd one out

a) Xanthine oxidase

b) NADH dehydrogenase –Q reductase

c) Succinate dehydrogenase

d) Cytochrome c                                                               

Q.4-All except one are incorrect about oxidases-

a) Oxidases catalyze reactions involving hydrogen peroxide.

b) Oxidases catalyze reaction using oxygen as a hydrogen acceptor

c) Oxidases catalyze  reactions using Niacin as coenzyme

d) Oxidases catalyze reactions of direct incorporation of oxygen in to the substrate

Q.5-All of the following are NAD+ requiring enzymes except one –

a) Acyl co A dehydrogenase

b) Glyceraldehyde-3-P dehydrogenase

c) Pyruvate dehydrogenase complex

d) Malate dehydrogenase

Q.6-One out of the following enzymes can utilize both NAD+ and NADP+as a coenzyme

a) Aldehyde dehydrogenase

b) Alcohol dehydrogenase

c) Glutamate dehydrogenase

d) Glycerol-3-P dehydrogenase

Q.7-Which out of the following components is not a haemo protein?

a) Catalase

b) Peroxidase

c) Ubiquinone

d) Cytochrome c                                                              

Q.8-Dioxygenases catalyze the incorporation of both atoms of oxygen in to the substrate. Which out of the following is a dioxygenase?

a) Tryptophan pyrrolase

b) Lactate dehydrogenase

c) Cytochrome oxidase

d) L- amino acid oxidase                                                  

Q.9 -Which of the components of electron transport chain does not contain Iron sulfur center?

a) NADH dehydrogenase complex

b) Cytochrome a-a3 oxidase

c) Succinate dehydrogenase

d) Cytochrome bc1-c reductase                                        

Q.10-A child has accidentally ingested a chemical and has presented with high fever.The chemical is known to affect ATP formation in electron transport chain,which out of the following could cause the similar manifestations

a) Cyanide

b) Malonate

c) 2,4 dinitrophenol

d) Rotenone                                                                    

Q.11-A 32- year female working in a laboratory consumed cyanide and was rushed to hospital. She was declared dead upon reaching the hospital. Cyanide is a known inhibitor of Electron Transport chain (ETC). Which complex of ETC might have been inhibited?

a) Complex I

b) Complex II

c) Complex III

d) Complex IV        

Q.12-The enzymes of mitochondrial matrix include all except

a) Enzymes of fatty acid oxidation

b) Creatine kinase

c) Enzymes of TCA cycle

d) Pyruvate dehydrogenase complex

Q.13-Patients with inherited defects of mitochondria involving components of the respiratory chain and oxidative phosphorylation present with all except

a) Myopathy

b) Lactic acidosis

c) Encephalopathy

d) Hepatomegaly

Q.14-The inner mitochondrial membrane is rich in which of the following phospholipids?

a) Cardiolipin

b) Lecithin

c) Cephalin

d) None of the above                                                       

Q.15-All are true about ATP synthase complex, except

a) F1projects into the inter membranous space

b) Fspans the membrane and forms a proton channel.

c) Fis inhibited by Oligomycin

d) F1contains the phosphorylation mechanism                     

Q.16-The energy yield during the conversion of succinate to Fumarate is-

a) 2ATP

b) 1ATP

c) 3ATP

d) No ATP                                                                     

Q.17-Which of the following best describes the biochemical basis of hyperthermia associated with Aspirin toxicity

a) Increased fatty acid oxidation

b) Increased muscular activity

c) Elevated consumption of ATP to support muscle contraction

d) Uncoupling of oxidative phosphorylation                       

Q.18-The electron flow from complex I to complex III is through

a) Cytochrome c

b) Ubiquinone

c) Complex II

d) Complex IV

Q.19-Which of the following best describes the toxicity associated with Atractyloside

a) Acts as an inhibitor of ETC

b) Acts as an uncoupler

c) Acts as an inhibitor of ATP/ADP transporter

d) Inactivates ATP synthase complex

Q.20- For each H2O molecule formed in ETC around ———– protons are pumped into inter membranous space

a) 4

b) 2

c) 10

d) None of the above

Q.21- Which one of the following enzymes catalyzes substrate level phosphorylation in TCA cycle

a) Malate dehydrogenase

b) Succinate Thiokinase

c) Succinate dehydrogenase

d) Alpha keto glutarate dehydrogenase complex

Q.22- 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

Q.23- One out of the following is an inhibitor of complex I

a) Rotenone

b) H2S

c) BAL

d) CN

Q.24- Which out of the following statements concerning the components of electron transport chain is true?

a) Oxygen directly oxidizes Cytochrome c

b) Succinate dehydrogenase directly reduces Cytochrome c

c) All of the components are embedded in the inner mitochondrial membrane

d) Cyanide does not inhibit proton pumping but inhibits ETC

Q.25- The free energy released during the transport of a pair of electrons in electron transport chain is

a) 7.3 Kcal/mol

b) 52.6 Kcal/mol

c) 21.9 Kcal/mol

d) None of the above

Key to answers

1)- (c), 2)-(c), 3)- (d), 4)- (b), 5)- (a), 6)- (c), 7)- (c), 8)- (a), 9)- (b),10)- (c),11)- (d), 12)- (b),13)- (d),14)- (a),

15) (a), 16)- (a),17)- (d),18)- (b),19)- (c), 20)-(c), 21)- (b), 22)- (b), 23)- (a), 24)- (d), 25)- (b). 

 


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Case Study

A 30 -year- old patient reported to medical OPD complaining of generalized weakness, excessive perspiration, and high caloric intake without increase in body weight. From the age of seven she had suffered from profuse sweating that forced her to change clothes about ten times a day. To compensate for the loss of fluids she had to drink many liters of water every day. Her calorie intake was about 3500 calories a day, approximately the need for a manual laborer, despite a body weight of only 40 kilograms.

There was no sign of any other disease. The BMR of the patient was excessively elevated but the thyroid hormones (T3 and T4) were in normal concentrations. Laboratory tests showed small deviations from the normal picture, with the exception of her blood volume, which was about twice as large as normal.

What is the probable diagnosis?

Case details The patient is probably suffering from Luft’s syndrome, also called Hypermetabolic mitochondrial syndrome.  

All the symptoms like high caloric intake but failure to gain weight, excessive perspiration, excessive thirst etc indicate a state of severe hyper metabolism of non thyroid origin (since thyroid hormones -T3 and T4 are normal). The defect lies in the maintenance of mitochondrial respiratory control.

Mitochondria play a major role in human energy dynamics (ATP synthesis and related reactions), and a functional deficit may be expected to result in diminished supply of high energy phosphate bonds. In mitochondria, electron transfer is coupled to oxidative phosphorylation via a proton gradient, and the energy released from oxidation is transferred to the ATP synthase trapping system. A class of compounds called uncouplers can block electron-transfer- linked phosphorylation at any of the three stages (energy production, transfer, or trapping). Redox reactions release free energy (delta G negative), such energy may be dissipated as heat, or be trapped as a proton gradient. Whether energy is dissipated or trapped depends on the characteristics of biologic membranes rather than on the reaction itself. Any substance that allows protons to leak across membranes will play an uncoupling role, since it will block the transfer of energy between electron flow and ATP synthesis.

More than 90% of the oxygen used in the human body is utilized by mitochondrial cytochrome oxidase, which transfers four electrons into an oxygen molecule to produce two molecules of water.

Deficits in mitochondrial function arise by two mechanisms: (1) a block in electron flow or ATP synthesis which results in lactic acidosis; and (2)uncoupling electron flow from ATP synthesis (Luft syndrome) in which oxidation occurs at a rapid rate but without a concomitant increase in ATP synthesis (energy released is dissipated as heat).

In Luft syndrome the uncoupling is caused by abnormality in the mitochondrial membrane. Electron transport is only loosely coupled to ATP production, thus the extra energy evolves in the form of heat.

Thus this syndrome is characterized by

·       Severe hyper metabolism,

·       Heat intolerance,

·       Profuse perspiration,

·       Polyphaga,

·       Polydipsia without polyuria,

·      Muscular wasting and weakness,

·      Absent deep reflexes, and

·      Resting tachycardia.

·   At the cellular level, the mitochondria respire wildly and waste the excess energy as heat, elevation of body temperature up to 38.4 °C.

·     There is progressive weight loss despite increased food intake.

·     Thyroid function is normal.

·      Onset is in childhood.

·     The rise in BMR is due to partial uncoupling of oxidative phosphorylation. Since there is less ATP production and the extra energy is lost in the form of heat , all the metabolic processes are stimulated. Due to high BMR only there is failure to put on weight despite a good diet.

Laboratory Investigations

Routine investigations can be undertaken for differential diagnosis. BMR, Thyroid function tests , blood glucose etc can be measured to rule out the presence of other conditions with similar symptoms.

Treatment

There is no permanent cure for this disease. Symptomatic treatment can only be given. Vitamin therapy in the form of Vitamins C, K, and E, along with coenzyme Q10 and a high-caloric diet is recommended.   Protection from heat is advised.

 

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Q.1- Out of the following respiratory chain components indicate which  one is a mobile carriers of electrons?
a) Cytochrome oxidase
b) NADH-Q reductase
c) Ubiquinone
d) Succinate dehydrogenase
Q.2- Choose the incorrect statement about redox potential
a) The redox potential of a system (E0)is usually compared with the potential of the hydrogen electrode
b) The components of electron transport chain are organized in terms of their redox potential.
c) NADH/NAD+ redox pair has the least redox potential of -0.42 volts
d) Oxygen/H2O redox pair has the highest redox potential of +.82 volts.
Q.3- All are flavoproteins except one, choose the odd one out-
a) Xanthine oxidase
b) NADH dehydrogenase –Q reductase
c) Succinate dehydrogenase
d) Cytochrome c
Q.4-All except one are incorrect about oxidases-
a) Oxidases catalyze reactions involving hydrogen peroxide.
b) Oxidases catalyze reaction using oxygen as a hydrogen acceptor
c) Oxidases catalyze  reactions using Niacin as coenzyme
d) Oxidases catalyze reactions of direct incorporation of oxygen in to the substrate.
Q.5- All of the followings are NAD+requiring enzymes except one –
a) Acyl co A dehydrogenase
b) Glyceraldehyde-3-P dehydrogenase
c) Pyruvate dehydrogenase complex
d) Malate dehydrogenase
Q.6- One out of the following enzymes can utilize  both NAD+ or NADP+ as a coenzyme-
a) Aldehyde dehydrogenase
b) Alcohol dehydrogenase
c) Glutamate dehydrogenase
d) Glycerol-3-P dehydrogenase
Q.7- Which out of the following components is not a haemo protein?
a) Catalase
b) Peroxidase
c) Ubiquinone
d) Cytochrome c
Q.8-Dioxygenases catalyze the incorporation of both atoms of oxygen in to the substrate. Which out of the following is a dioxygenase?
a) Tryptophan pyrrolase
b) Lactate dehydrogenase
c) Cytochrome oxidase
d) L- amino acid oxidase
Q.9- Which of the components of electron transport chain does not contain Iron sulfur center?
a) NADH dehydrogenase complex
b) Cytochrome a-a3 oxidase
c) Succinate dehydrogenase
d) Cytochrome bc1-c reductase
Q.10- A child has accidentally ingested a chemical and has presented with high fever. The chemical is known to affect ATP formation in electron transport chain , which out of the followings could cause the similar manifestations
a) Cyanide
b) Malonate
c) 2,4 dinitrophenol
d) Rotenone
Q.11- A 32- year female working in a laboratory consumed cyanide and was rushed to hospital .She was declared dead upon reaching  the hospital.Cyanide is a known inhibitor of Electron Transport chain (ETC). Which complex of ETC might have been inhibited?
a) Complex I
b) Complex II
c) Complex III
d) Complex IV
Q.12-The enzymes of mitochondrial matrix include all except-
a) Enzymes of fatty acid oxidation
b) Creatine kinase
c) Enzymes of TCA cycle
d) Pyruvate dehydrogenase complex
Q.13-Patients with inherited defects of mitochondria involving components of the respiratory chain and oxidative phosphorylation present with all except-
a) Myopathy
b) Lactic acidosis
c) Encephalopathy
d) Hepatomegaly
Q.14- The inner mitochondrial membrane is rich in which of the following phospholipids?
a) Cardiolipin
b) Lecithin
c) Cephalin
d) None of the above
Q.15- All are true about ATP synthase complex, except-
a) F1 projects into the inter membranous space
b) F0 spans the membrane and forms a proton channel.
c) F0 is inhibited by Oligomycin
d) F1contains thephosphorylation mechanism
Q.16- The energy yield during the conversion of succinate to Fumarate is-
a) 2 ATP
b) 1 ATP
c) 3 ATP
d) No ATP
Q.17- Which of the following best describes the biochemical basis of hyperthermia associated with Aspirin toxicity
a) Increased fatty acid oxidation
b) Increased muscular activity
c) Elevated consumption of ATP to support muscle contraction
d) Uncoupling of oxidative phosphorylation
Q.18-The electron flow from complex I to complex III is through
a) Cytochrome c
b) Ubiquinone
c) Complex II
d) Complex IV
Q.19-Which of the followings best describes the toxicity associated with Atractyloside
a) Acts as an inhibitor of ETC
b) Acts as an uncoupler
c) Acts as an inhibitor of ATP/ADP transporter
d) Inactivates ATP synthase complex
Q.20- For each H2O molecule formed in ETC around ———–  protons are pumped into
inter membranous space
a) 4
b) 2
c) 10
d) none of the above
Q.21- Which one of the following enzymes catalyzes substrate level phosphorylation in TCA cycle
a) Malate dehydrogenase
b) Succinate Thiokinase
c) Succinate dehydrogenase
d) Alpha keto glutarate dehydrogenase complex
Q.22- 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
Q.23- One out of the following is an inhibitor of complex I
a) Rotenone
b) H2S
c) BAL
d) CN
Q.24- Which out of the following statements concerning the components of electron transport chain is true?
a) Oxygen directly oxidizes Cytochromec
b) Succinate dehydrogenase directly reduces Cytochrome c
c) All of the components are embedded in the inner mitochondrial membrane
d) Cyanide does not inhibit proton pumping but inhibits ETC
Q.25- The free energy released during the transport of a pair of electrons in electron transport chain is-
a) 7.3 Kcal/mol
b) 52.6 Kcal/mol
c) 21.9 Kcal/mol
d) None of the above
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1.   Give a brief account of  each of the followings
a)    P:O ratio
b)    Redox Potential
c)    ATP Synthase complex
d)    Mitochondrial shuttles
e)    Site specific inhibitors of electron transport chain
f)     Uncouplers  of oxidative phosphorylation
2.   Give a diagrammatic representation of Electron Transport chain showing the flow of electrons, sites of ATP formation and the sites of inhibitors of Electron transport chain.
3.   Show by means of a diagram the process of ATP formation by oxidative phosphorylation.
4.   Show by means of diagrams the mechanisms of transfer of NADH from cytoplasm to mitochondria .
5.   Differentiate between substrate level phosphorylation and oxidative phosphorylation.
6.   Explain why rotenone which inhibits mitochondrial oxidation and Uncouplers (such as 2,4 DNP) are lethal to cells and organisms 
7.   For each of the followings, write at least three reactions  where they are required as co enzymes. (a) NAD+ (b) FAD
8.   Define the P:O ratio in mitochondrial oxidation. Briefly explain the different values obtained with succinate as compared to malate as the substrate.
9.   Explain briefly why the oxidation of succinate to fumarate yields only 2 mol ATP per mol of succinate oxidized but oxidation of malate to oxaloacetate yields 3 mol ATP per mol of malate oxidized ?
10.  Describe briefly the structure of the mitochondrial Fl-F0 ATPase (ATP synthase complex) and explain how it functions in the synthesis of ATP?
11.   Explain the reason that the toxic dosage of Aspirin can cause hyperthermia?
12.  Explain why the complete oxidation of I mol of Glucose in muscles yields 36 mol ATP while in liver this amounts to   38 mol ATP?
13.   What is the biological advantage of uncoupling of oxidative phosphorylation?
14.   What is the biochemical basis of uncoupling of oxidative phosphorylation in brown adipose tissue?
15.  Catalase and Peroxidase, both act as scavengers for decomposition of H2O2, how do they differ in their actions?
16.  Give a brief account of inhibitors of ATP synthesis. 
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