Bio-energetics and biochemistry information

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1. EMB-RCG Bio-energetics and oxidative phosphor ylation

2. Energy EMB-RCG

3. The ultimate source of energy is the food that we consume Carbohydrates, lipids and proteins present in food provide us energy These are present in food in the form of large complex molecules EMB-RCG

4. The carbon atoms are oxidized to carbon dioxide Hydrogen atoms are transferred to coenzymes e.g. NAD + , FMN, FAD etc EMB-RCG

5. The reduced coenzymes transfer the hydrogen atoms to the mitochondrial respiratory chain wherein these are oxidized to water The energy released during this oxidation is used to phosphorylate adenosine diphosphate (ADP) to adenosine triphosphate (ATP) EMB-RCG

6. Oxidation coupled with phosphorylation of ADP is known as oxidative phosphorylation Oxidative phosphorylation, thus, is the mechanism by which the energy present in various nutrients is captured in an easily utilizable form EMB-RCG

7. High-energy phosphates The energy released during the oxidation of monosaccharides, fatty acids and amino acids may not be required immediately Therefore, there must be some way of storing the energy so that it may be readily available when needed The energy released during catabolism is captured in the form of a group of compounds known as “ high-energy phosphates”

8. The most important high-energy phosphate is ATP Hydrolysis of ATP into ADP and Pi liberates 7.3 kcal of energy per mol Hydrolysis of ADP into AMP and Pi also releases nearly the same amount of energy

9. However, hydrolysis of AMP into adenosine and Pi liberates much less energy (3.4 kcal/mol) This difference is because of the nature of bonds by which phosphate is attached The third phosphate is attached to the second, and the second to the first, by acid anhydride bond The first phosphate is attached to ribose by an ester bond

10. Adenosine triphosphate O

11. Hydrolysis of acid anhydride bonds releases much more energy than the hydrolysis of ester bonds Lipmann suggested a curved line (~) to denote a high-energy bond Thus, ATP may be represented as : Adenosine— P ~ P ~ P

12. Compound D G o Compound D G o Phosphoenol pyruvate – 14.8 AMP ( ® Adenosine + Pi) – 3.4 Carbamoyl phosphate – 12.3 Glucose-1-phosphate – 5.0 1,3-Biphosphoglycerate – 11.8 Fructose-6-phosphate – 3.8 Creatine phosphate – 10.3 Glucose-6-phosphate – 3.3 ATP ( ® ADP + Pi) – 7.3 Glycerol-3-phosphate – 2.2 Standard free energy ( D G o ) of hydrolysis of some important organic phosphates (kcal/mol)* *These are the values of D G o obtained in standard laboratory conditions of 1M reactant concentration at pH 7.0 at 25°C; values obtained in living cells ( D G’ o ) are different as the reactant concentrations , pH and temperature are different EMB-RCG

13. The organic phosphates which liberate 6 kcal/mol or more on hydrolysis of the phosphate group are known as high-energy phosphates Those organic phosphates which release less than 6 kcal/mol on hydrolysis of the phosphate group are known as low-energy phosphates EMB-RCG

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15. The compounds having D G o above that of ATP can transfer their phosphate group to ADP forming ATP The compounds having D G o below that of ATP can not transfer their phosphate groups to ADP On the contrary, ATP can transfer a phosphate group to glucose, fructose, glycerol etc forming their respective phosphates EMB-RCG

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17. Besides high-energy phosphates, some sulphur compounds having thio-ester bonds are also high-energy compounds In acetyl CoA, succinyl CoA, acyl CoA etc, CoA is attached by a high-energy thio-ester bond R - C ~S - CoA O EMB-RCG

18. Oxidation and reduction Oxidation was defined in the past as addition of oxygen to or removal of hydrogen from a substance The reverse was termed as reduction These definitions have now been supplanted by a more comprehensive concept EMB-RCG

19. Oxidation is now defined as removal of electrons and reduction as addition of electrons The electron being removed or added may be a free electron or it may be associated with a proton as in a hydrogen atom EMB-RCG

20. Our earliest concepts of oxidation in living beings (biological oxidation) originated from the work of Lavoisier He proposed that respiration in animals is an oxidative process in which atmospheric oxygen is used to oxidize carbon atoms to carbon dioxide EMB-RCG

21. Pasteur showed later that the presence of oxygen is not essential for oxidation , and that living organisms can oxidize substrates even in the absence of oxygen Wieland proposed that biological oxidation occurred by dehydrogenation of activated substrates EMB-RCG

22. With the discovery of cytochromes by Keilin, it became clear that the substrates are dehydrogenated The reducing equivalents ( H or e – ) are taken up by cytochromes They are finally transferred to oxygen in the presence of cytochrome oxidase (Warburg’s enzyme) to be converted into water EMB-RCG

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24. The sequence of carriers in the respiratory chain 1 3

25. Redox potential A reactant which can undergo reduction and oxidation can exist in the reduced form and the oxidized form The reduced and the oxidized forms of the reactant constitute a redox couple Every redox couple has a redox potential which is a measure of the affinity of the reactant for electrons EMB-RCG

26. A redox couple having a high redox potential has a high affinity for electrons It can readily accept electrons from a redox couple having a lower redox potential The redox potential of a reactant can be measured in the laboratory EMB-RCG

27. Measurement of redox potential The reduced and the oxidised forms of the reactant at 1M concentration each are taken in a sample cell A 1M solution of hydrogen ions in equilibrium with hydrogen gas is taken in a reference cell The two are connected by an agar bridge through which electrons can flow EMB-RCG

28. Measurement of redox potential (Contd) Electrodes dipping in each solution are connected to a voltmeter The potential difference between the two solutions is the redox potential of the reactant EMB-RCG

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30. For biological systems, the redox potential (E o ) is measured at pH 7.0, and is denoted by E’ o The components of the respiratory chain are arranged in the order of increasing redox potential The electrons move from a relatively electronegative component to a relatively electropositive component at every site EMB-RCG

31. Redox potential (E’ o ) of carriers in the respiratory chain Redox couple E´ o (volts) Redox couple E´ o (volts ) 2H + - H 2 – 0.42 Cyt c 1 (Fe +3 ) - Cyt c 1 ( Fe +2 ) + 0.22 NAD + - NADH – 0.32 Cyt c (Fe +3 ) - Cyt c ( Fe +2 ) + 0.25 FAD - FADH 2 – 0.22 Cyt a (Fe +3 ) - Cyt a ( Fe +2 ) + 0.29 Cyt b(Fe +3 )- Cyt b(Fe +2 ) – 0.08 Cyt a 3 ( Fe +3 ) - Cyt a 3 ( Fe +2 ) + 0.38 CoQ - CoQH 2 + 0.04 ½ O 2 - H 2 O +0.82 EMB-RCG

32. Enzymes involved in biological oxidation The enzymes concerned with biological oxidation are oxido-reductases They can be sub-divided into: Oxidases Dehydrogenases Hydroperoxidases Oxygenases EMB-RCG

33. Oxidases These enzymes remove hydrogen from a substrate and transfer it to oxygen forming water or hydrogen peroxide The enzymes forming water are metalloenzymes that usually contain copper e.g. cytochrome oxidase and tyrosinase The general reaction catalysed by these enzymes is: AH + ½ O A + H O 2 2 2

34. The enzymes forming hydrogen peroxide are flavoproteins containing FMN or FAD e.g. L-amino acid oxidase (containing FMN) and xanthine oxidase (containing FAD ) These enzymes catalyse the general reaction: AH 2 + O 2 A + H 2 O 2 EMB-RCG

35. Dehydrogenases These are conjugated proteins containing nicotinamide nucleotides or flavin nucleotides or iron-porphyrin as the prosthetic group They remove hydrogen from a substrate, and transfer it to another substrate The prosthetic group acts as carrier of hydrogen atoms

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37. Examples of dehydrogenases are : Those containing NAD - Lactate dehydrogenase , malate dehydrogenase etc Those containing NADP - Glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydro- genase etc Those containing flavin nucleotides - Succinate dehydrogenase, acyl CoA dehydrogenase etc Those containing iron-porphyrin - Cytochrome a , cytochrome b etc

38. Hydroperoxidases These enzymes convert hydrogen peroxide into water, and protect the tissues against the toxic effects of hydrogen peroxide These include: A. Peroxidase – It catalyses the reaction: H 2 O 2 + AH 2 ® A + 2 H 2 O B. Catalase – It catalyses the reaction: 2 H 2 O 2 ® O 2 + 2 H 2 O

39. Oxygenases These enzymes incorporate oxygen into a substrate They can be sub-divided into: Di-oxygenases Mono-oxygenases

40. Di-oxygenases These enzymes catalyse the incorporation of both the atoms of oxygen molecule into the substrate A + O 2 AO 2 Examples include homogentisate oxidase and tryptophan pyrrolase

41. Mono-oxygenases These enzymes incorporate one atom of the oxygen molecule into the substrate The other atom of oxygen oxidises another reduced substrate to water AH + O 2 + BH 2 A–OH + B + H 2 O

42. These enzymes are also known as hydroxylases or mixed function oxidases They are used to metabolize xenobiotics (foreign compounds), for example drugs like morphine, phenobarbitone, rifampicin etc They also catalyse hydroxylation of endogenous substrates e.g . phenylalanine , tryptophan , steroids , cholecalciferol etc

43. Two slightly different hydroxylase systems are present in the cells: Microsomal hydroxylase system Mitochondrial hydroxylase system

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46. NADPH - Cytochrome P - 450 reductase Cytochrome P - 450 (mono- oxygenase ) Microsomal hydroxylase system EMB-RCG

47. The reaction catalysed by microsomal hydroxylase system O 2 5 CYP-Fe | | AH O ++ 2 AH CYP-Fe | | AH O +++ – 2 CYP-Fe | | AH O +++ – – 2 CYP-Fe +++ 4 3 4 3 CYP-Fe | AH ++ CYP-Fe | AH +++ 4 6 6 3 3 5 4 NADPH + H + NADP + FAD FADH 2 4 FMNH 2 FMN 4 e – 2H + e – A-OH+H 2 O 4

48. Mitochondral hydroxylase system Present in the inner membrane of mitochondria Catalyses hydroxylation of endogenous substrates The reaction catalysed by this system is basically similar to the microsomal hydroxylation reaction

49. The enzyme is NADPH:adrenodoxin reductase (a flavoprotein containing FAD) instead of NADPH:cytochrome P-450 reductase The reducing equivalents accepted by FAD are transferred to cytochrome P-450 via adrenodoxin ( iron-sulphur protein)

50. Iron-sulphur centres Adrenodoxin possesses an iron-sulphur centre having catalytic activity

51. NADPH - Adrenodoxin reductase Cytochrome P - 450 (mono-oxygenase) Iron-sulphur protein NADPH + H + NADP + AH + O 2 A - OH + H 2 O Mitochondral hydroxylase system FAD F +++ FeS EMB-RCG

52. Metabolism of superoxide r adicals Hydrogen peroxide is continuously formed in the body by the action of flavoprotein oxidases It was believed in the past that the toxicity of molecular oxygen is solely due to its conversion into hydrogen peroxide

53. It was shown later that toxic effects of oxygen are also due to its conversion into free radicals or superoxide radicals (O 2 – ) Free radicals, once formed, can start a chain reaction forming more free radicals These highly reactive radicals can damage nucleic acids, proteins and lipids, specially unsaturated fatty acids

54. Several diseases are now believed to occur due to damage caused by free radicals Superoxide radicals can be formed due to incomplete oxidation of reduced flavoprotein oxidases FpH 2 + O 2 FpH + H + + O 2 -

55. Several endogenous and exogenous compounds act as anti-oxidants They either prevent the formation of free radicals or detoxify the already formed free radicals

56. The endogenous anti-oxidants include: Superoxide dismutase Glutathione peroxidase Catalase Cytochrome c Uric acid

57. The exogenous (nutritional) anti-oxidants include: Vitamin A Vitamin C Vitamin E Selenium Carotene Several plant pigments found in vegetables and fruits

58. One mechanisms by which the superoxide radicals can be detoxified is: O 2 – + Fe +++ (Cyt c) O 2 + Fe ++ (Reduced cyt c) Cytochrome c accepts an electron from the superoxide radical

59. In another mechanism, superoxide dismutase converts superoxide radicals into hydrogen peroxide which can, then , be converted into water by catalase : 2 O 2 - + 2 H + Superoxide dismutase H 2 O 2 + O 2 Superoxide dismutase is a widely distributed metalloenzyme

60. The flavoprotein oxidases which produce hydrogen peroxide are present in sub-cellular organelles called peroxisomes Catalase, which detoxifies hydrogen peroxide, is also present in peroxisomes This protects the cell against the toxic effects of hydrogen peroxide

61. Peroxisomes are congenitally absent in Zellweger’s syndrome This results in extensive damage to brain, liver and kidneys

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65. In the glycolytic pathway, oxidation of glyceraldehyde-3-phosphate is linked with introduction of a high-energy phosphate in the product

66. In the next reaction, the high energy phosphate is transferred to ADP forming ATP

67. H COOH C O P CH 2 OH COOH H 2 O C O P CH 2 CH 2 COOH OH C ADP ATP Enolase Enol pyruvate Phosphoenol pyruvate 2-Phospho- glycerate Pyruvate kinase The high-energy phosphate is transferred to ADP in the next reaction In another reaction of same pathway, energy released during dehydration of 2-phospho- glycerate converts the low-energy phosphate bond of the substrate into a high-energy bond

68. In one reaction of citric acid cycle , the energy released during oxidation of a - ketoglutarate is used to form a high-energy bond between succinate and CoA In the next reaction, CoA is split off and the energy released is used to phosphorylate GDP to GTP CH — COOH 2 | CH — C — COOH 2 CH — COOH 2 CH — COOH 2 | | O CH —COOH 2 NADH + H + CO + 2 GDP + Pi GTP + CoA–SH a -Ketoglutarate dehydrogenase Succinate thiokinase CH — C ~ S — CoA 2 O a -Ketoglutarate Succinyl CoA Succinate NAD + CoA—SH +

69. CH 2 — COOH | CH 2 — C — COOH CH 2 — COOH CH 2 — COOH | | CH 2 — COOH NADH + H + CO + 2 GDP + Pi GTP + CoA–SH a -Ketoglutarate dehydrogenase Succinate thiokinase CH 2 — C ~ S — CoA O a -Ketoglutarate Succinyl CoA Succinate NAD + CoA—SH + O In one reaction of citric acid cycle, the energy released during oxidation of a - ketoglutarate is used to form a high-energy bond between succinate and CoA In the next reaction, CoA is split off and the energy released is used to phosphorylate GDP to GTP

70. The energy captured by substrate-linked oxidative phosphorylation is only a fraction of the energy present in a nutrient For example, complete oxidation of one molecule of glucose via glycolysis and citric acid cycle phosphorylates 38 molecules of ADP Of these, only six are phosphorylated at the substrate level The rest of the energy is captured in the respiratory chain

71. Oxidative p hosphorylation at the level of respiratory c hain Energy-rich nutrients like glucose, fatty acids and amino acids are oxidized stepwise by a series of reactions in various metabolic pathways In many reactions, reducing equivalents are removed from the substrates, and are taken up by coenzymes like NAD and FAD

72. The reduced coenzymes transfer the reducing equivalents to respiratory chain Oxidation of the reducing equivalents in the respiratory chain is coupled with the phosphorylation of ADP to ATP This is the most important mechanism for capturing the energy present in various nutrients As the respiratory chain is located in the mitochondria, the mitochondria have been described as the power house of the cells

73. Most of the substrates transfer the reducing equivalents to NAD Reduced NAD transfers them to a flavoprotein containing FMN and iron-sulphur (FeS) centre Reduced flavoprotein transfers the reducing equivalents to coenzyme Q (ubiquinone) Transport of reducing equivalents in respiratory chain

74. Coenzyme Q is a fat-soluble compound resembling vitamin K in structure It contains 6-10 isoprenoid units (n=6-10), and can be reduced to ubiquinol

75. Reduced coenzyme Q transfers the reducing equivalents to cytochrome b which is associated with iron-sulphur protein The subsequent carriers, cytochromes c 1 , c and a, are typical iron-porphyrin proteins These differ from each other in their protein portions and in the side chains attached to the porphyrin nucleus The cytochromes transport electrons

76. Prosthetic group of cytochrome a

77. Prosthetic group of cytochrome b Prosthetic group of cytochrome c

78. The electrons are taken up and transferred by the iron portion of the cytochromes, which can oscillate between Fe +++ and Fe ++ forms The last cytochrome (cytochrome a 3 ) is an oxidase which catalyses the transfer of reducing equivalents to oxygen forming water

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80. Components of the respiratory chain do not function as discrete carriers of reducing equivalents They are organized into four complexes each of which acts as a specific oxidoreductase Coenzyme Q and cytochrome c are not parts of any complex , and are not fixed in the inner mitochondrial membrane The other components are fixed in the membrane

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82. Complex I, II, III and IV in respiratory chain EMB-RCG

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84. Complex I acts as NADH : ubiquinone oxidoreductase, and transfers reducing equivalents from NADH to CoQ

85. Complex II acts as succinate : ubiquinone oxidoreductase, and transfers reducing equivalents from succinate to CoQ

86. Complex III acts as ubiquinol : ferricytochrome c oxidoreductase, and transfers reducing equivalents from reduced CoQ to cytochrome c

87. Complex IV acts as ferrocytochrome c : oxygen oxidoreductase,and transfers reducing equivalents from reduced cytochrome c to oxygen

88. The proximal end of the respiratory chain has a negative redox potential while the distal end has a positive redox potential As the electrons move from a relatively electronegative component to a relatively electropositive component, energy is released

89. The quantum of energy released at any site is proportional to the difference in the redox potentials of the component donating the reducing equivalents and the component accepting the reducing equivalents Thus, different quanta of energy are released at different sites

90. Hydrolysis of the terminal phosphate of ATP releases 7.3 kcal of energy per mol in standard laboratory conditions But in physiological conditions prevailing in living cells , the energy required for phosphorylation of ADP to ATP is about 10 kcal/mol

91. In the respiratory chain, the quantum of energy released exceeds 10 kcal/mol at those sites where the difference between the redox potentials of the donor and the acceptor of reducing equivalents is 0.3 volts or more ADP is phosphorylated at these sites There are three such sites in the respiratory chain

92. Site I is between NAD and CoQ Site II is between Co Q and cytochrome c Site III is between cytochrome c and oxygen These sites correspond to complexes I, III and IV respectively in the respiratory chain

93. All the substrates undergoing dehydrogenation do not transfer the reducing equivalents to NAD Reducing equivalents are accepted by a carrier having a redox potential just above that of the substrate

94. The substrates that transfer reducing equivalents to NAD, e.g. isocitrate, malate, glutamate etc, can phosphorylate three molecules of ADP The substrates that transfer reducing equivalents to FAD (e.g. succinate, glycerol-3- phosphate, acyl CoA etc) phosphorylate only two molecules of ADP as Site I is bypassed

95. The ratio of ADP molecules phosphorylated to the number of oxygen atoms reduced is known as P:O ratio The P:O ratio is three when the reducing equivalents are accepted by NAD, and is two when the reducing equivalents are accepted by FAD

96. Mechanism of oxidative phosphorylation Mechanism by which oxidation of reducing equi- valents and phosphorylation of ADP are coupled has remained unclear for long Several hypotheses have been advanced to explain the mechanism of this coupling of which the chemiosmotic hypothesis is the most plausible

97. Chemiosmotic hypothesis Proposed by Mitchell Inner mitochondrial membrane is impermeable to protons(H + ) Energy released during transport of electrons in the respiratory chain is used to actively eject H + from the matrix of mitochondria This ejection establishes an electrochemical gradient across the membrane

98. Complexes I, III and IV in the respiratory chain act as proton pumps ejecting hydrogen ions from the mitochondrial matrix to the inter-membrane space Succinate Fumarate

99. The concentration of H + on the outer side becomes higher as compared to the inner side The outer side also becomes electropositive as compared to the inner side

100. This electrochemical gradient increases up to a certain limit When this limit is reached, the hydrogen ions re-enter the matrix releasing energy

101. Re-entry of protons releases energy

102. The energy released during influx of protons is used to activate a membrane-bound enzyme, vectorial ATP synthetase which converts ADP and Pi into ATP

103. Efraim Racker showed that: Vectorial ATP synthetase is made up of F and F 1 components F component is embedded in the inner mitochondrial membrane F 1 component projects into the matrix

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105. F 1 component F component Inner mitochondrial membrane EMB-RCG

106. F component acts as a channel for the passage of hydrogen ions F 1 component possesses ATP synthetase activity This activity is switched on when the hydrogen ions pass through the F component

107. John Walker deciphered the genes encoding the F and F 1 components The F component is made up of a, b and c subunits The F 1 component is made up of a, b, g, d and e subunits ( a 3 b 3 gde ) The subunits of F and F 1 components of are encoded by a cluster of genes called the unc operon

108. The subunits of F and F 1 components of vectorial ATP synthetase EMB-RCG

109. This form readily combines with ADP by a high-energy bond Water is formed and inorganic phosphate is converted into a highly reactive form Two hydrogen ions combine with two electrons and one oxygen atom of inorganic phosphate When hydrogen ions flow back into the matrix: EMB-RCG

110. O || P — O O — P — O — P — O — A –– O || O || || O | | – O – O H 2 O H 2 O 3 O || 2H + – O — P — O – – O — P — O — P — O — A O || O || | – O | | – O – O Pi ADP 2H + 4 4 4 O || – O — P — O — P — O — P — O — A O || O || | – O | | – O – O ATP

111. Paul Boyer has shown that: F 1 complex has three sites having specific conformations – O site (open site) , L site (loose-binding site) and T site (tight-binding site) Each site is made up of one a and one b subunit The three sites are inter-convertible In the absence of an electrochemical gradient , equal amounts of ATP and ADP&Pi are bound to the T and L sites respectively

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113. Conversion of T site into O site releases the bound ATP Conversion of L site into T site converts the bound ADP & Pi into ATP Another pair of ADP & Pi enters the new L site, and the cycle is repeated EMB-RCG

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115. The inter-conversion of sites occurs because of rotation of a and b subunits of F 1 component The rotation occurs in steps of 120 o

116. There is strong experimental evidence in support of chemiosmotic hypothesis Building up of H + gradient is the basic premise of the hypothesis It has been shown that if mitochondria are bathed in a fluid having a relatively high H + concentration, phosphorylation occurs even in the absence of oxidation

117. The hypothesis envisages a membrane-bound vectorial ATP synthetase It has been shown that disruption of the mitochondrial membrane leads to loss of ATP synthetase activity

118. The P:H + and H + : O ratios of various substrates are also in agreement with the experimental evidence Thus, the chemiosmotic hypothesis has now become the accepted theory to explain the mechanism of oxidative phosphorylation in the respiratory chain

119. Inhibitors of oxidative p hosphorylation Certain agents are known to inhibit oxidative phosphorylation at specific sites in the respiratory chain Amobarbitone, rotenone and piericidin A which inhibit oxidative phosphorylation at site I have now been shown to inhibit the oxidoreductase activity of complex I

120. Dimercaprol and antimycin inhibit the oxidoreductase activity of complex III Hydrogen sulphide, carbon monoxide and cyanide inhibit the oxidoreductase activity of complex IV When oxidation is inhibited, phosphorylation also can not occur

121. Oligomycin inhibits oxidative phosphorylation at all the sites It binds to and inhibits F component of ATP synthetase In fact, the subscript ‘o’ derives from the tendency of this component to bind oligomycin

122. Inhibitors of Oxidative Phosphorylation

123. Uncouplers of oxidative phosphorylation Certain agents, e.g. dinitrophenol, dinitrocresol and dicoumarol, uncouple oxidation and phosphorylation In their presence, phosphorylation is inhibited but oxidation goes on

124. It has been shown that the uncouplers make the inner mitochondrial membrane freely permeable to hydrogen ions This does not allow the electrochemical gradient to build up and, therefore, ATP can not be synthesized even though oxidation is going on

125. An endogenous uncoupler Thermogenin is a protein present in brown adipose tissue Brown adipose tissue is rich in mitrochondia Thermogenin is present in inner mitochondrial membrane It acts as a channel for entry of hydrogen ions into mitochondria

126. Thermogenin Inner mitochondrial membrane Inter- membrane space Mitochondrial matrix H +

127. As hydrogen ion gradient can not build up, phosphorylation does not occur Oxidation occurs in brown adipose tissue without generation of ATP resulting in production of heat Brown adipose tissue is present in significant amount in infancy and decreases with age

128. Regulation of oxidative p hosphorylation Oxidative phosphorylation in the respiratory chain results in consumption of oxygen This is also known as tissue respiration When oxidizable substrates and oxygen are available, the rate of tissue respiration is regulated mainly by the concentration of ADP

129. Increased utilization of ATP raises the concentration of ADP This stimulates tissue respiration resulting in increased phosphorylation of ADP into ATP

130. Oxidation of extra-mitochondrial NADH Most of the NADH is produced in mitochondria But some NADH is produced in cytosol also Mitochondrial membrane is impermeable to NADH Special mechanisms are required to transport NADH from cytosol into mitochondria

131. Two important mechanisms are: Malate shuttle Glycerophosphate shuttle EMB-RCG

132. Glycerophosphate shuttle Cytosolic NADH is used to reduce dihydroxy- acetone phosphate to glycerol-3-phosphate which goes into mitochondria as the mitochondrial membrane is permeable to it In the mitochondria, glycerol-3-phosphate is oxidized to dihydroxyacetone phosphate which comes our into the cytosol In the mitochondrial reaction, the hydrogen atoms are transferred to FAD

133. Therefore, only two ADP molecules are phosphorylated when FADH 2 is oxidised in the respiratory chain

134. Malate shuttle This mechanism is quantitatively more significant Cytosolic malate dehydrogenase (MDH) transfers reducing equivalents from NADH to oxaloacetate forming NAD and malate Malate goes into mitochondria, and is oxidized to oxaloacetate by mitochondrial MDH

135. NAD accepts the reducing equivalents Hence there is no loss of energy But mitochondrial membrane is not permeable to oxaloacetate For transporting oxaloacetate back to cytosol, a special mechanism is required

136. Mitochondrial glutamate oxaloacetate trans- aminase (GOT) transfers an amino group from glutamate to oxaloacetate forming a -keto- glutarate and aspartate These two come out of the mitochondria, and are reconverted into glutamate and oxaloacetate by cytosolic GOT Glutamate , then, goes back into mitochondria

137. The net result is that the cytosolic NADH has been converted into NAD, and the mitochondrial NAD has been converted into NADH 4 3 4 3 Cytosol Mitochondrial Matrix NADH+H + MDH Oxalo- acetate Oxalo- acetate Aspartate Aspartate NADH+H + 3 3 4 3 3 3 GOT Glutamate NAD + NAD + Malate a -Keto- glutarate a -Keto- glutarate 4 3 3 3 MDH Glutamate Malate 4 GOT 5

138. Transport across m itochondrial m embrane The mitochondrial membrane is selectively permeable Small uncharged molecules and mono- carboxylic acids can pass through the membrane easily Special transport mechanisms are required to transport di-carboxylic acids, tri-carboxylic acids and amino acids as the membrane is impermeable to these

139. These transport mechanisms may operate as : Symports - Two compounds traversing the membrane in the same direction Antiports - Two compounds traversing the membrane in opposite directions

140. Transport of pyruvate and H + into mitochondria is an example of a symport Membrane Mitochondrial matrix Cytosol Pyruvate Pyruvate H + H +

141. Memb Matrix Cytosol ADP ADP ATP ATP Malate a- Ketoglutarate Malate Malate Citrate + H + + + Glutamate Glutamate Aspartate Aspartate Some important antiports Malate a- Ketoglutarate Citrate + H +