1. الدكتور الصيدلاني احمد يحيى دلال باشي استاذ في الكيمياء الحياتيه الطبية مقرر فرع الكيمياء الحياتيه

2. Bioenergetics and Biological Oxidation

3. Titles of subjects in lecture 1 Bioenergetics and Biological1. Bioenergetics and Biological Oxidation Oxidation 2. Adinosine triphosphate (ATP)2. Adinosine triphosphate (ATP) 3. Coupled reactions3. Coupled reactions

4. Bioenergetics and Biological Oxidation Bioenergetics is the study of energy changes or thermodynamic accompanying biochemical reactions, which takes place inside the body. Bioenergetics is the study of energy changes or thermodynamic accompanying biochemical reactions, which takes place inside the body. The reactions in general are accompanied by liberation of energy, they may require energy, or neither gives nor requires energy. Energy is necessary for biological system to perform the different biochemical and physiological activities. This energy can be obtained from the breakdown of different nutritional substances (Fuel compounds. lipids, carbohydrates and proteins). Energy is necessary for biological system to perform the different biochemical and physiological activities. This energy can be obtained from the breakdown of different nutritional substances (Fuel compounds. lipids, carbohydrates and proteins). This explain why in certain conditions e.g. during starvation for several days or weeks may end with death and this can be accounted due to depletion of energy from all stores or reserves of energy inside the body.

5. In certain other conditions when there are defects in energy or other metabolic problems may result in certain metabolic disorders as e.g. in case of Kwashiorkor and Marasmus in which there are defect in energy supply and quantity and types of proteins. In certain other conditions when there are defects in energy or other metabolic problems may result in certain metabolic disorders as e.g. in case of Kwashiorkor and Marasmus in which there are defect in energy supply and quantity and types of proteins. On the other hand increased energy intake more than body requirement will result in obesity. On the other hand increased energy intake more than body requirement will result in obesity. However, the energy release is regulated by thyroid hormones. However, the energy release is regulated by thyroid hormones. In normal condition, there is a fine control between two processes these are energy requirement and energy releasing. In normal condition, there is a fine control between two processes these are energy requirement and energy releasing. This control or link is very important in bioenergetics as it occur through ATP (Adenosine triphosphate) which is made up of 3 parts This control or link is very important in bioenergetics as it occur through ATP (Adenosine triphosphate) which is made up of 3 parts 1)A nitrogenous base (Adenine) 2) A sugar (Ribose) & 3) 3 Phosphoric acid molecules as shown.

6. The two outer phosphate atoms are very important in the bioenergetics as the bonds in these phosphates are high energy ( ~ ). On hydrolysis of ATP, it gives adenosine diphosphate + Pi, in addition to energy. On hydrolysis of ATP, it gives adenosine diphosphate + Pi, in addition to energy. ATP

7. ATP ADP+Pi+ Energy(≈7Kcal) This means it release the terminal inorganic phosphate and so it is converted to ADP+Pi. This hydrolysis usually accompanied by liberation of energy or heat of about -7 Kcal (-30Kj)/ mol. (- ve charge mean available energy while +ve charge mean required energy. Under certain conditions further hydrolysis of ADP may occur resulting in the production of AMP + Pi and this accompanied by liberation of almost the same quantity of energy. ADP AMP + Pi + (≈7Kcal) The phosphate atom in AMP is not considered as high-energy phosphate and so on hydrolysis will not produce energy. The phosphate atom in AMP is not considered as high-energy phosphate and so on hydrolysis will not produce energy.

8. Titles of subjects in lecture 2 Coupled Reactions: Biological Oxidation: Respiratory chain

9. Coupled Reactions: The high-energy reaction (~), some times called energy favorable reaction or exergonic reaction is usually coupled to another reaction e.g. if a compound A is converted to B in certain biological system. If A is stable compound then B cannot appear in the medium unless a catalyst** is present (Enzyme*) so an enzyme (E) is required The high-energy reaction (~), some times called energy favorable reaction or exergonic reaction is usually coupled to another reaction e.g. if a compound A is converted to B in certain biological system. If A is stable compound then B cannot appear in the medium unless a catalyst** is present (Enzyme*) so an enzyme (E) is required E A B E A B

10. * Enzyme: It is a catalyst in biological reactions. It is a protein in nature. * Enzyme: It is a catalyst in biological reactions. It is a protein in nature. ** Catalyst: It is certain substance, which help in increasing the speed of the reaction. It may shear in the reaction and may suffer from physical changes but it return to its original form at the end of the reaction.) ** Catalyst: It is certain substance, which help in increasing the speed of the reaction. It may shear in the reaction and may suffer from physical changes but it return to its original form at the end of the reaction.) If this reaction is energy favorable then this reaction require another reaction to capture heat or energy from the first reaction, otherwise it will dissipate. These reactions are called coupled reactions and can be facilitated by the enzymes. Therefore, the enzyme may be designed for the presence of ADP & Pi, which result in the formation of ATP. E ADP+Pi ATP

11. This reaction is a non favorable reaction (endergonic) and require energy, if we collect both reactions in one equation A + ADP + Pi E B + ATP A + ADP + Pi E B + ATP Another example if a compound C E D C E D Where this reaction require an enzyme. If this reaction is non-favorable then it will require an energy and so the enzyme in this case will provide ATP which will be hydrolyses to ADP+Pi in addition to energy. ATP ADP + Pi ATP ADP + Pi When we collect both reactions: C + ATP E D + ADP + Pi C + ATP E D + ADP + Pi We can summarize the 2 reactions as follows:

12. A D A D ADP + Pi ATP B C B C When A is changed to B energy is produced and captured by ADP+Pi and when C changed to D it require energy and can be obtained from ATP.

13. However, all human and animals activities require energy, which can be available from the breakdown of different fuel compounds in stepwise breakdown (Metabolism) in the cells and any defect or fault will lead to certain disease due to abnormal metabolic disorder. Exergonic and endergonic reactions are also terms used to describe the energy favorable and energy required reactions respectively. Exergonic or energy favorable reactions supply energy for the normal biochemical and physiological functions of the body e.g. muscle contraction, nerve excitation, active transport, synthetic reactions and so on. Exergonic or energy favorable reactions supply energy for the normal biochemical and physiological functions of the body e.g. muscle contraction, nerve excitation, active transport, synthetic reactions and so on.

14. Biological Oxidation: About 1780 Lavoisier concluded that combustion processes must takes place in animal’s organism. Since then biological oxidation has often been compared to combustion. Indeed there is no difference in the product in both cases are CO2 + H2O + Energy. Oxidation is defined today very generally as a loss of electrons (e). Oxidation of molecular hydrogen can therefore be formulated as follows H 2 - 2e - 2H + ……1 H 2 - 2e - 2H + ……1 An oxidizing agent must accept the electrons. If we use e.g. a ferric salt, the equation becomes: H2 + 2 Fe +++ = 2H + + 2Fe ++ ………. 2 Molecular oxygen can act as an oxidizing agent similarly by picking up either 2 or 4 electrons:

15. +2H + +2H + O2 + 2 e - = O 2 -2 H 2 O 2 …3 +4H+ +4H+ O2 + 4 e - = 2O -2 2H 2 O ……4 O2 + 4 e - = 2O -2 2H 2 O ……4 Therefore, Oxidation is the removal of electrons and reduction is the gain of electrons. Oxidation is always accompanied by reduction of an electron acceptor. Also, dehydrogenation means oxidation of a compound. In chemical oxidation O2 can oxidizes the compound directly while in biological system O2 is not directly oxidizes the compound but it shear in oxidation at the final stage, certain enzymes and coenzymes* are responsible for oxidation instead of oxygen. *Coenzyme: It is a prosthetic group in the enzyme molecule it is non-protein organic substance, which inter really in the reaction but cannot catalyse the reaction without the enzyme. They usually contain certain member of the B-complex vitamin.

16. The sequence of the enzymes and carriers responsible for the transport of reducing equivalent (e) from substrate* to molecular oxygen is known as respiratory chain or electron transport chain or mitochondrial transport system, which is imbedded in the inner membrane of the mitochondria. *Substrate is the substance upon which the enzymes act on. This will carry the electrons on a series of carriers and when pass from one carrier to another the first is oxidized and the next is reduced and the final one is oxygen. At the end, the final compound is converted to energy in addition to H2O + CO2, which are excreted to the outside. Many enzymes and coenzymes participate in biological oxidation some of them are members of the respiratory chain in the mitochondria. These are called oxido-reductases. They include:

17. 1. Oxidases 2. Dehydrogenases: a. aerobic dehydrogenases a. aerobic dehydrogenases b. Anaerobic dehydrogenases: b. Anaerobic dehydrogenases: dependent on Nicotiamide (NAD&NADP) dependent on Nicotiamide (NAD&NADP) dependent on flavine (FAD&FMN) dependent on flavine (FAD&FMN) 3. Cytochromes 4. Hydroperoxidases: Peroxidase Catalases Catalases 5. Oxygenases: Mono Di Di 6. CoQ (ubiquinone)

18. Titles of subjects in lecture 3 1. Oxidases 2. Dehydrogenases: a. aerobic dehydrogenases a. aerobic dehydrogenases b. Anaerobic dehydrogenases: b. Anaerobic dehydrogenases: dependent on Nicotiamide dependent on Nicotiamide (NAD&NADP) (NAD&NADP) dependent on flavine (FAD&FMN) dependent on flavine (FAD&FMN)

19. Oxidases: These are enzymes involved in oxidation-reduction reaction in biological system. They act by the removal of hydrogen from one substrate to another and the substrate will be oxidized. They use only oxygen as hydrogen acceptor.

20. Oxidases usually come in the last stage in biological oxidation. Water is always the product of this reaction. Example of oxidases is the cytochrome oxidase or called (a3) although this enzyme as a compound is cytochrome but the mechanism of action is belonging to oxidases. Dehydrogenases: a) Aerobic dehydrogenases : These enzymes catalyses the removal of hydrogen from certain substrates and can use oxygen or other artificial compounds as hydrogen acceptor e.g. of artificial compounds is methylene blue.

22. In this reaction, H2O2 always is the product. Aerobic dehydrogenases are usually contain flavoprotein as coenzyme i.e. coenzyme containing FAD or FMN. FAD: Flavine adnine dinucleotide (Containing riboflavin or vit B2) FMN: Flavine mononucleotide. Riboflavine or Vit B2 : Consist of ribitol and flavine, available in all vegetables and milk, deficiency cause inflammation of the tongue and angles of the mouth (Chilosis).

26. b) Anaerobic dehydrogenases : These are enzymes catalysis the oxidation of substrate but can not use oxygen as hydrogen acceptor instead they utilize other metabolite as hydrogen acceptor. They are divided into: These are enzymes catalysis the oxidation of substrate but can not use oxygen as hydrogen acceptor instead they utilize other metabolite as hydrogen acceptor. They are divided into: 1. Anaerobic dehydrogenases dependent on NAD or NADP as coenzyme :(They were called Co I and Co II respectively) NAD: Nicotinamide Adenine dinucleotide NADP: Nicotinamide Adenine dinucleotide Phosphate. A large number of enzymes are classified on this class. They contain in their structure the vitamin niacin or vit B3.

29. Niacine or Vit B3 (Nicotinic acid) is a non toxic part of a toxic alkaloid present in tobacco (nicotin). Niacine present in Meat, Fish, Whole & Enriched Grains, Beans, Nuts, milk and leafy vegetables. Diffeciency causes Pellagra( or 3 d) Niacine or Vit B3 (Nicotinic acid) is a non toxic part of a toxic alkaloid present in tobacco (nicotin). Niacine present in Meat, Fish, Whole & Enriched Grains, Beans, Nuts, milk and leafy vegetables. Diffeciency causes Pellagra( or 3 d) (Dermatitis, Dementia, and diarrhea). NADP usually used during synthetic reactions particularly the fatty acids and steroids synthesis and they have important role in HMP shunt. NADP usually used during synthetic reactions particularly the fatty acids and steroids synthesis and they have important role in HMP shunt. While NAD may participate mainly in glycolysis, in citric acid cycle and in other metabolic pathways. NAD is also a member of the respiratory chain of the mitochondria. The active part of NAD that participates in the reaction is the nicotinamide. While NAD may participate mainly in glycolysis, in citric acid cycle and in other metabolic pathways. NAD is also a member of the respiratory chain of the mitochondria. The active part of NAD that participates in the reaction is the nicotinamide.

30. NAD is the direct oxidant it will be reduced to NADH+H + and when NADH + H + accumulate, it can be reoxidised to NAD

31. 2- Anaerobic dehydrogenases dependent on Riboflavin: 2- Anaerobic dehydrogenases dependent on Riboflavin: The active part is riboflavin or vit B2 e.g. of this coenzyme is FAD and FMN. They have important role as hydrogen carrier in certain oxidation-reduction reactions. It is possible that the reduced form of NAD (NADH2) can be reoxidise by dehydrogenases using FAD and so another oxidation- reduction process will occur as follows:

32. Titles of subjects in lecture 4 3. Cytochromes 4. Hydroperoxidases: Peroxidase 4. Hydroperoxidases: Peroxidase Catalases Catalases 5. Oxygenases: Mono Di Di 6. CoQ (ubiquinone)

33. Cytochromes: They are group of enzymes, which are haeme protein (Contain 4 pyrrol rings). Pyrrol ring Porphyrin Pyrrol ring Porphyrin

34. They are members of respiratory chain. They function as carries of electrons from CoQ to cytochrome oxidase. The cytochromes contain iron as the active part in the center of porphyrin (Cytochrome). The Fe atom will oscillate from ferric to ferrous (Fe +3 and Fe +2 ) during oxidation and reduction. When these enzymes catalyse the oxidation, the metabolite will be oxidized and Fe atom will be reduced. At least five cytochromes were isolated in the respiratory chain of the mitochondria. These are cytochrome b, c1, c, a & a3.

35. Hydroperoxidases: They are group of enzymes act mainly using hydrogen peroxide (H2O2) as a substrate. They are divided into 2 groups: a) Peroxidases b) Catalases Peroxidases: They are usually present in the lymphocytes, platelets and monocytes. Their function is to convert H 2 O2 to water.

36. 2H2O2 2H2O + O2 2H2O2 2H2O + O2 If H2O2 accumulate in the cells may causes damage to the cells and this group of enzymes by oxidation change H2O2 to water. If H2O2 accumulate in the cells may causes damage to the cells and this group of enzymes by oxidation change H2O2 to water.Catalases: It is a haeme protein containing four haeme groups. They are found in blood and the liver. Their function is the destruction of H2O2 to water. It is a haeme protein containing four haeme groups. They are found in blood and the liver. Their function is the destruction of H2O2 to water.

37. Oxygenases: Oxygenases: They are group of enzymes catalysis the incorporation of oxygen into substrate molecule. They are divided into: They are group of enzymes catalysis the incorporation of oxygen into substrate molecule. They are divided into: a) Dioxygenases: They incorporate the two atoms of oxygen into the substrate. They incorporate the two atoms of oxygen into the substrate. A + O2 AO2 A + O2 AO2 e.g.L-tryptophane dioxygenase e.g.L-tryptophane dioxygenase

38. b. Monooxygenases: These enzymes incorporate one atom of oxygen into substrate molecule. The other oxygen atom is reduced to water e.g. some enzymes, which are involved in steroid synthesis; also, some enzymes involved into metabolism of some drugs are belonging to monooxygenases. They are usually present in the microsomes of the liver together with cytochrome p450 and cytochrome b5. These enzymes incorporate one atom of oxygen into substrate molecule. The other oxygen atom is reduced to water e.g. some enzymes, which are involved in steroid synthesis; also, some enzymes involved into metabolism of some drugs are belonging to monooxygenases. They are usually present in the microsomes of the liver together with cytochrome p450 and cytochrome b5. Super oxide ( ): This result due to partial reduction of oxygen during redox reaction (Oxidation-reduction reaction). The super oxide anion has the property of oxidizing and reducing agent. Usually the result of super oxide anion is H2O2, which is a toxic product and elimination, occur under the effect of catalases and peroxidases.

39. CoQ or ubiquinone: This coenzyme is present in the inner mitochondrial membrane. It is a member of the respiratory chain. It is a lipid compound and has the property of electron carrier. It is quinone derivative when oxidized called quinone and when reduced called hydroquinone. This coenzyme is present in the inner mitochondrial membrane. It is a member of the respiratory chain. It is a lipid compound and has the property of electron carrier. It is quinone derivative when oxidized called quinone and when reduced called hydroquinone.

40. Respiration and respiratory chain: The mitochondrion in each cell is regarded as the power house. Inside the mitochondria, there are series of carrier, which carry the electrons (also called reducing equivalents). This system is called respiratory chain or mitochondrial transport system or electron transport chain. This chain or system is imbedded in the inner mitochondrial membrane arranged in certain manner through increasing redox potential. The reason for this arrangement may be due to that NAD which is the first member of the respiratory chain which accept H from the substrate specific dehydrogenase ) if it is directly react with oxygen then large amount of energy will be produced. This energy may be larger than the ability of a single coupling reaction and so energy may dissipate. Instead it is subdivided into small individual energy so NADH2 does not react directly with oxygen but in a series of intermediate steps with the possibility of coupling each step with other reaction thus keeping much of the energy formed. As the energy of biological system is in the form of ATP so the respiratory chain coupled with phosphorylation i.e. with the formation of ATP from ADP+Pi and the whole process is called oxidative phosphorylation.

41. Sequence of Redox system in the respiratory chain: The arrangement of component enzymes and coenzymes in the respiratory chain depend on the redox potential. The most negative potential is that of NAD so it is the first member of the RC, which can receive H from the substrate and become reduced. The arrangement of component enzymes and coenzymes in the respiratory chain depend on the redox potential. The most negative potential is that of NAD so it is the first member of the RC, which can receive H from the substrate and become reduced. The hydrogen is then transferred to the next member of the RC (FP) and so the first member (NADH2) is reoxidised and the second one is reduced and become FPH2 then the H will be transferred to the third (CoQ) and so flavoprotein will be reoxidised. The hydrogen is then transferred to the next member of the RC (FP) and so the first member (NADH2) is reoxidised and the second one is reduced and become FPH2 then the H will be transferred to the third (CoQ) and so flavoprotein will be reoxidised. CoQ is also a point or a site for the entry of hydrogen from the dehydrogenation of succinate and fatty acids. The next member of the RC does the reoxidation of CoQ, which is the cytochrome system that acts by change of valence of iron. From this point, only electrons are transferred. CoQ is also a point or a site for the entry of hydrogen from the dehydrogenation of succinate and fatty acids. The next member of the RC does the reoxidation of CoQ, which is the cytochrome system that acts by change of valence of iron. From this point, only electrons are transferred.

42. The cytochromes are arranged also according to their redox potential in the sequence shown where cytochrome aa3 transfer the electrons to oxygen, which immediately pick up 2H + to produce water. The cytochromes are arranged also according to their redox potential in the sequence shown where cytochrome aa3 transfer the electrons to oxygen, which immediately pick up 2H + to produce water. Fatty Acids and Fatty Acids and Substrate succinate Specific NAD FP CoQ Cytochrome b Dehydrogenase Ratenon Amytal Amytal Cytochrome c1 Cytochrome c Cytochrome c1 Cytochrome c Antimycin A CN Antimycin A CN Cytochrome aa3 O2 Cytochrome aa3 O2 CO CO Certain inhibitors like Ratenon and Amytal, antimycin A, Cyanide and CO can block the redox at the sites shown.

43. Redox occurs in the Mitochondria: Any oxidation-reduction reaction is accompanied by transfer of electrons between the two systems. This happen in the mitochondria as pair of electrons are given to NAD which is the first member of the RC from different intermediate like glutamate, pyrovate, α-keto acids by different dehydrogenases systems. This pair of electrons then flows from the first member of the RC (NAD) to the second member (FP) and so on until the final component of the RC giving its pair of (e) to oxygen forming water. This pair of electrons then flows from the first member of the RC (NAD) to the second member (FP) and so on until the final component of the RC giving its pair of (e) to oxygen forming water.

44. There are certain intermediate which cannot give their pair of electrons to the first member (NAD) instead they give it to CoQ and this is due to charge on the compound. There are certain intermediate which cannot give their pair of electrons to the first member (NAD) instead they give it to CoQ and this is due to charge on the compound. When the electrons go to CoQ, they also flow in the same direction of the chain and finally reach oxygen to produce water and energy. When the electrons go to CoQ, they also flow in the same direction of the chain and finally reach oxygen to produce water and energy. Therefore, the electrons can pass to the RC either through NAD or through CoQ. It is clear that the transfer of electrons from one member to another in the RC is an oxidation-reduction process. The following scheme shows the redox in the mitochondria. The following scheme shows the redox in the mitochondria.

45. Sites of ATP production: Each pair of electrons when it enter the chain from the beginning till react with oxygen then one high energy product (ATP) is produced in 3 sites between NADH2 and FP, CoQ or ubiquinone and cytochrome b and Cytochrome aa3 and O2. NAD  FP  CoQ  Cyto b  Cyto C1  Cyto C  Cyto aa3  O2 site I ADP + Pi  ATP site II ADP + Pi  ATP site III ADP + Pi  ATP

46. Therefore 3 molecules of ATP are produced from the cycle when started from the beginning (NAD) to the end. While when the electrons enter the RC from CoQ then 2 ATP are formed as it miss or bypass the first site of synthesis of ATP. Therefore 3 molecules of ATP are produced from the cycle when started from the beginning (NAD) to the end. While when the electrons enter the RC from CoQ then 2 ATP are formed as it miss or bypass the first site of synthesis of ATP. Oxidative Phosphorylation: The importance of RC is because free energy of oxidation of some of the individual steps are trapped or stored in the form of ATP using ADP+Pi in each site. This process is termed oxidative phosphorylation or RC phosphorylation. The importance of RC is because free energy of oxidation of some of the individual steps are trapped or stored in the form of ATP using ADP+Pi in each site. This process is termed oxidative phosphorylation or RC phosphorylation. It is found that 1 ATP can arise for each pair of electrons (or hydrogen) transferred at each of 3 sites between NADH2 and FP, ubiquinone and Cyto b and Cyto aa3 and oxygen. It is found that 1 ATP can arise for each pair of electrons (or hydrogen) transferred at each of 3 sites between NADH2 and FP, ubiquinone and Cyto b and Cyto aa3 and oxygen.

47. Control of Respiration: Normally the redox of RC and RC phosphorylation are coupled tightly. Normally the redox of RC and RC phosphorylation are coupled tightly. Redox reaction cannot proceed unless ADP is phosphorylated. ADP thus becomes the limiting factor of the over all reactions including oxygen uptake. This is called control of respiration by ADP. When large amount of ATP are consumed as happen during muscle contraction then the accumulation of ADP stimulate respiration and so the greater the supply of ADP the greater the rate of transport of electrons to oxygen or respiration.

48. Uncouplers and inhibitors of phosphorylation: 2, 4-Dinitrophenol, certain derivatives of phenyl hydrazones and other compounds are called uncouplers of oxidative phosphorylation because they may stimulate respiration of mitochondria; at the same time prevent formation of ATP. Other inhibitors like Oligomycin prevent both respiration and phosphorylation. Also it is found that when protein is stripped off the phosphorylating electron transport particles then phosphorylation stop, while the addition of protein will restore phosphorylation.

49. Mechanism of phosphorylation reaction of RC: The mechanism of phosphorylation of RC is unclear. Two theories are now acceptable: a. Chemical Coupling: The following diagram may illustrate the theory:

50. Initially the redox reaction is coupled with the formation of an energy–rich product for the three phosphorylation sites C1~X, C2~X, & C3~X. These high energy compounds by ATPase (Y) can form X~Y. This compound is a high energy product which can react with inorganic phosphate to form ~P. This ~P is transported from X to ADP with the formation of ATP. ATP goes to the cytoplasm by translocase system in the inner mitochondrial membrane.

51. b. The Chemiosmatic theory: Oxidation of components in the respiratory chain generate 2H + in each coupling sites which are ejected or exported outside of a coupling membrane in the mitochondria (from matrix to the intermembranal space). The electrochemical potential difference resulting from the asymmetric distribution of the hydrogen ions is used to derive back H + to the matrix through ATPase. The electrochemical potential difference resulting from the asymmetric distribution of the hydrogen ions is used to derive back H + to the matrix through ATPase. During passage through ATPase and in presence of ADP& Pi then ATP is formed. It is found that for each 2H + exported to any coupling site then one ATP is formed.

52. The high energy compound, ATP speaks: I am the energy currency of the cell! Continuous consumption and regeneration is my role; Without me, all biochemical functions come to a standstill; Existence of life is unimaginable without my will ATP