1. Introduction to metabolism. Specific and general pathways of carbohydrates, lipids and proteins metabolism. Oxidative decarboxylation of pyruvate. Krebs cycle. Bioenergetic processes: biological oxidation.Substrate and oxidative phosphorilation. The tiny hummingbirds can store enough fuel to fly a distance of 500 miles without resting. This achievement is possible because of the ability to convert fuels into the cellular energy currency, ATP.

2. Metabolism - the entire network of chemical reactions carried out by living cells. Metabolism also includes coordination, regulation and energy requirement. Metabolites - small molecule intermediates in the degradation and synthesis of polymers Most organism use the same general pathway for extraction and utilization of energy. All living organisms are divided into two major classes: Autotrophs – can use atmospheric carbon dioxide as a sole source of carbon for the synthesis of macromolecules. Autotrophs use the sun energy for biosynthetic purposes. Heterotrophs – obtain energy by ingesting complex carbon- containing compounds. Heterotrophs are divided into aerobs and anaerobs.

3. Common features of organisms 1. Organisms or cells maintain specific internal concentrations of inorganic ions, metabolites and enzymes 2. Organisms extract energy from external sources to drive energy-consuming reactions 3. Organisms grow and reproduce according to instructions encoded in the genetic material 4. Organisms respond to environmental influences 5. Cells are not static, and cell components are continually synthesized and degraded (i.e. undergo turnover)

4. (a) Linear (b) Cyclic (c) Spiral pathway (fatty acid biosynthesis) A sequence of reactions that has a specific purpose (for instance: degradation of glucose, synthesis of fatty acids) is called metabolic pathway. Metabolic pathway may be:

5. Catabolic reactions - degrade molecules to create smaller molecules and energy Anabolic reactions - synthesize molecules for cell maintenance, growth and reproduction Metabolic pathways can be grouped into two paths – catabolism and anabolism Catabolism is characterized by oxidation reactions and by release of free energy which is transformed to ATP. Anabolism is characterized by reduction reactions and by utilization of energy accumulated in ATP molecules. Catabolism and anabolism are tightly linked together by their coordinated energy requirements: catabolic processes release the energy from food and collect it in the ATP; anabolic processes use the free energy stored in ATP to perform work.

6. Anabolism and catabolism are coupled by energy

7. Stages of metabolism Catabolism Stage I. Breakdown of macromolecules (proteins, carbohydrates and lipids to respective building blocks. Stage II. Amino acids, fatty acids and glucose are oxidized to common metabolite (acetyl CoA) Stage III. Acetyl CoA is oxidized in citric acid cycle to CO 2 and water. As result reduced cofactor, NADH 2 and FADH 2, are formed which give up their electrons. Electrons are transported via the tissue respiration chain and released energy is coupled directly to ATP synthesis.

8. Glycerol Catabolism

9. Catabolism is characterized by convergence of three major routs toward a final common pathway. Different proteins, fats and carbohydrates enter the same pathway – tricarboxylic acid cycle. Anabolism can also be divided into stages, however the anabolic pathways are characterized by divergence. Monosaccharide synthesis begin with CO 2, oxaloacetate, pyruvate or lactate. Amino acids are synthesized from acetyl CoA, pyruvate or keto acids of Krebs cycle. Fatty acids are constructed from acetyl CoA. On the next stage monosaccharides, amino acids and fatty acids are used for the synthesis of polysaccharides, proteins and fats.

10. Compartmentation of metabolic processes

11. OXIDATIVE DECARBOXYLATION OF PYRUVATE Matrix of the mitochondria contains pyruvate dehydrogenase complex

12. The fate of glucose molecule in the cell Glucose Glucose-6- phosphate Pyruvate Glycogen Ribose, NADPH Pentose phosphate pathway Synthesis of glycogen Degradation of glycogen Glycolysis Gluconeogenesis LactateEthanol Acetyl Co A

13. Only about 7 % of the total potential energy present in glucose is released in glycolysis. Glycolysis is preliminary phase, preparing glucose for entry into aerobic metabolism. Pyruvate formed in the aerobic conditions undergoes conversion to acetyl CoA by pyruvate dehydrogenase complex. Pyruvate dehydrogenase complex is a bridge between glycolysis and aerobic metabolism – citric acid cycle. Pyruvate dehydrogenase complex and enzymes of cytric acid cycle are located in the matrix of mitochondria. OXIDATIVE DECARBOXYLATION OF PYRUVATE

14. Pyruvate translocase, protein embedded into the inner membrane, transports pyruvate from the intermembrane space into the matrix in symport with H + and exchange (antiport) for OH -. Entry of Pyruvate into the Mitochondrion Pyruvate freely diffuses through the outer membrane of mitochon- dria through the channels formed by transmembrane proteins porins.

15. Pyruvate dehydrogenase complex (PDH complex) is a multienzyme complex containing 3 enzymes, 5 coenzymes and other proteins. Conversion of Pyruvate to Acetyl CoA Pyruvate dehydrogenase complex is giant, with molecular mass ranging from 4 to 10 million daltons. Electron micrograph of the pyruvate dehydrogenase complex from E. coli.

16. Enzymes: E1 = pyruvate dehydrogenase E2 = dihydrolipoyl acetyltransferase E3 = dihydrolipoyl dehydrogenase Coenzymes: TPP (thiamine pyrophosphate), lipoamide, HS-CoA, FAD+, NAD+. TPP is a prosthetic group of E1; lipoamide is a prosthetic group of E2; and FAD is a prosthetic group of E3. The building block of TPP is vitamin B 1 (thiamin); NAD – vitamin B 5 (nicotinamide); FAD – vitamin B 2 (riboflavin), HS-CoA – vitamin B 3 (pantothenic acid), lipoamide – lipoic acid

17. Overall reaction of pyruvate dehydrogenase complex Pyruvate dehydrogenase complex is a classic example of multienzyme complex The oxidative decarboxylation of pyruvate catalized by pyruvate dehydrogenase complex occurs in five steps.

18. Glucose Glucose-6- phosphate Pyruvate Glycogen Ribose, NADPH Pentose phosphate pathway Synthesis of glycogen Degradation of glycogen Glycolysis Gluconeogenesis LactateEthanol Acetyl Co A Fatty Acids Amino Acids The citric acid cycle is the final common pathway for the oxidation of fuel molecules — amino acids, fatty acids, and carbohydrates. Most fuel molecules enter the cycle as acetyl coenzyme A.

19. Names: The Citric Acid Cycle Tricarboxylic Acid Cycle Krebs Cycle In eukaryotes the reactions of the citric acid cycle take place inside mitochondria Physiology or Medicine. Hans Adolf Krebs. Biochemist; born in Germany. Worked in Britain. His discovery in 1937 of the ‘Krebs cycle’ of chemical reactions was critical to the understanding of cell metabolism and earned him the 1953 Nobel Prize for Physiology or Medicine.

20. 1. Citrate Synthase Citrate formed from acetyl CoA and oxaloacetate Only cycle reaction with C-C bond formation Addition of C 2 unit (acetyl) to the keto double bond of C 4 acid, oxaloacetate, to produce C 6 compound, citrate citrate synthase

21. 2. Aconitase Elimination of H 2 O from citrate to form C=C bond of cis-aconitate Stereospecific addition of H 2 O to cis-aconitate to form isocitrate aconitase

22. 3. Isocitrate Dehydrogenase Oxidative decarboxylation of isocitrate to a-ketoglutarate (a metabolically irreversible reaction) One of four oxidation-reduction reactions of the cycle Hydride ion from the C-2 of isocitrate is transferred to NAD + to form NADH Oxalosuccinate is decarboxylated to a-ketoglutarate isocitrate dehydrogenase

23. 4. The  -Ketoglutarate Dehydrogenase Complex Similar to pyruvate dehydrogenase complex Same coenzymes, identical mechanisms E 1 - a-ketoglutarate dehydrogenase (with TPP) E 2 – dihydrolipoyl succinyltransferase (with flexible lipoamide prosthetic group) E 3 - dihydrolipoyl dehydrogenase (with FAD)  -ketoglutarate dehydrogenase

24. 5. Succinyl-CoA Synthetase Free energy in thioester bond of succinyl CoA is conserved as GTP or ATP in higher animals (or ATP in plants, some bacteria) Substrate level phosphorylation reaction HS- + GTP + ADP GDP + ATP Succinyl-CoA Synthetase

25. Complex of several polypeptides, an FAD prosthetic group and iron-sulfur clusters Embedded in the inner mitochondrial membrane Electrons are transferred from succinate to FAD and then to ubiquinone (Q) in electron transport chain Dehydrogenation is stereospecific; only the trans isomer is formed 6. The Succinate Dehydrogenase Complex Succinate Dehydrogenase

26. 7. Fumarase Stereospecific trans addition of water to the double bond of fumarate to form L-malate Only the L isomer of malate is formed Fumarase

27. 8. Malate Dehydrogenase Malate Dehydrogenase Malate is oxidized to form oxaloacetate.

28. Stoichiometry of the Citric Acid Cycle  Two carbon atoms enter the cycle in the form of acetyl CoA.  Two carbon atoms leave the cycle in the form of CO 2.  Four pairs of hydrogen atoms leave the cycle in four oxidation reactions (three molecules of NAD + one molecule of FAD are reduced).  One molecule of GTP, is formed.  Two molecules of water are consumed.  9 ATP (2.5 ATP per NADH, and 1.5 ATP per FADH 2 ) are produced during oxidative phosphorylation.  1 ATP is directly formed in the citric acid cycle.  1 acetyl CoA generates approximately 10 molecules of ATP.

29. Integration of metabolism. The citric acid cycle is amphibolic (both catabolic and anabolic). Functions of the Citric Acid Cycle The cycle is involved in the aerobic catabolism of carbohydrates, lipids and amino acids. Intermediates of the cycle are starting points for many anabolic reactions. Yields energy in the form of GTP (ATP). Yields reducing power in the form of NADH 2 and FADH 2.

30. Regulation of the Citric Acid Cycle Pathway controlled by: (1) Allosteric modulators (2) Covalent modification of cycle enzymes (3) Supply of acetyl CoA (pyruvate dehydrogenase complex) Three enzymes have regulatory properties - citrate synthase (is allosterically inhibited by NADH, ATP, succinyl CoA, citrate – feedback inhibition) - isocitrate dehydrogenase (allosteric effectors: (+) ADP; (-) NADH, ATP. Bacterial ICDH can be covalently modified by kinase/phosphatase) -  -ketoglutarate dehydrogenase complex (inhibition by ATP, succinyl CoA and NADH

31. NADH, ATP, succinyl CoA, citrate - Regulation of the citric acid cycle

32. Krebs Cycle is a Source of Biosynthetic Precursors Phosphoenol- pyruvate Glucose The citric acid cycle provides intermediates for biosyntheses

33. Reduced coenzymes NADH and FADH 2 are formed in matrix from: (1) Oxidative decarboxilation of pyruvate to acetyl CoA (2) Aerobic oxidation of acetyl CoA by the citric acid cycle (3) Oxidation of fatty acids and amino acids The NADH and FADH 2 are energy-rich molecules because each contains a pair of electrons having a high transfer potential.

34. The reduced and oxidized forms of NAD

35. The reduced and oxidized forms of FAD

36. Electrons of NADH or FADH 2 are used to reduce molecular oxygen to water. A large amount of free energy is liberated. The electrons from NADH and FADH 2 are not transported directly to O 2 but are transferred through series of electron carriers that undergo reversible reduction and oxidation.

37. The flow of electrons through carriers leads to the pumping of protons out of the mitochondrial matrix. The resulting distribution of protons generates a pH gradient and a transmembrane electrical potential that creates a protonmotive force.

38. ATP is synthesized when protons flow back to the mitochondrial matrix through an enzyme complex ATP synthase. The oxidation of fuels and the phosphorylation of ADP are coupled by a proton gradient across the inner mitochondrial membrane. Oxidative phosphorylation is the process in which ATP is formed as a result of the transfer of electrons from NADH or FADH 2 to O 2 by a series of electron carriers.

39. OXIDATIVE PHOSPHORYLATION IN EUKARYOTES TAKES PLACE IN MITOCHONDRIA Two membranes: outer membrane inner membrane (folded into cristae) Two compartments: (1) the intermembrane space (2) the matrix Inner mitochondrial membrane: Electron transport chain ATP synthase Mitochondrial matrix: Pyruvate dehydrogenase complex Citric acid cycle Fatty acid oxidation Location of mitochondrial complexes The outer membrane is permeable to small molecules and ions because it contains pore-forming protein (porin). The inner membrane is impermeable to ions and polar molecules. Contains transporters (translocases).

40. THE ELECTRON TRANSPORT CHAIN Series of enzyme complexes (electron carriers) embedded in the inner mitochondrial membrane, which oxidize NADH 2 and FADH 2 and transport electrons to oxygen is called respiratory electron-transport chain (ETC). The sequence of electron carriers in ETC cyt b NADH FMN Fe-S Co-Q Fe-S cyt c 1 cyt c cyt a cyt a 3 O 2 succinate FAD Fe-S

41. High-Energy Electrons: Redox Potentials and Free-Energy Changes In oxidative phosphorylation, the electron transfer potential of NADH or FADH 2 is converted into the phosphoryl transfer potential of ATP. Phosphoryl transfer potential is  G°' (energy released during the hydrolysis of activated phos-phate compound).  G°' for ATP = -7.3 kcal mol -1 Electron transfer potential is expressed as E' o, the (also called redox potential, reduction potential, or oxidation-reduction potential).

42. E' o (reduction potential) is a measure of how easily a compound can be reduced (how easily it can accept electron). All compounds are compared to reduction potential of hydrogen wich is 0.0 V. The larger the value of E' o of a carrier in ETC the better it functions as an electron acceptor (oxidizing factor). Electrons flow through the ETC components spontaneously in the direction of increasing reduction potentials. E' o of NADH = -0.32 volts (strong reducing agent) E' o of O 2 = +0.82 volts (strong oxidizing agent) cyt b NADH FMN Fe-S Co-Q Fe-S cyt c 1 cyt c cyt a cyt a 3 O 2 succinate FAD Fe-S

44. Important characteristic of ETC is the amount of energy released upon electron transfer from one carrier to another. This energy can be calculated using the formula:  G o ’=-nF  E’ o n – number of electrons transferred from one carrier to another; F – the Faraday constant (23.06 kcal/volt mol);  E’ o – the difference in reduction potential between two carriers. When two electrons pass from NADH to O 2 :  G o ’=-2*96,5*(+0,82-(-0,32)) = -52.6 kcal/mol

45. Components of electron- transport chain are arranged in the inner membrane of mitochondria in packages called respiratory assemblies (complexes). THE RESPIRATORY CHAIN CONSISTS OF FOUR COMPLEXES cyt b NADH FMN Fe-S Co-Q Fe-S cyt c 1 cyt c cyt a cyt a 3 O 2 succinate FAD Fe-S I III II IV I II III IV

47. The energy is released not in a single step of electron transfer but in incremental amount at each complex. 26.8 Energy released at three specific steps in the chain is collected in form of transmembrane proton gradient and used to drive the synthesis of ATP.

48. Complexes I-IV Mobile coenzymes: ubiquinone (Q) and cytochrome c serve as links between ETC complexes Complex IV reduces O 2 to water

49. Transfers electrons from NADH to Co Q (ubiquinone) Consist of: - enzyme NADH dehydrogenase (FMN - prosthetic group) - iron-sulfur clusters. NADH reduces FMN to FMNH 2. Electrons from FMNH 2 pass to a Fe-S clusters. Fe-S proteins convey electrons to ubiquinone. QH 2 is formed. Complex I (NADH-ubiquinone oxidoreductase) The flow of two electrons from NADH to coenzym Q leads to the pumping of four hydrogen ions out of the matrix.

50. matrix NADH-Q oxidoreductase - an enormous enzyme consisting of 34 polypeptide chains. L-shaped (horizontal arm lying in the membrane and a vertical arm that projects into the matrix). FMN NADH Iron ions in Fe-S complexes cycle between Fe 2+ or Fe 3+ states. Iron-sulfur clusters contains two or four iron ions and two or four inorganic sulfides. Clusters are coordinated by four cysteine residues. Fe-S

51. Complex II (succinate-ubiquinon oxidoreductase) Transfers electrons from succinate to Co Q. Form 1 consist of: - enzyme succinate dehydrogenase (FAD – prosthetic group) - iron-sulfur clusters. Succinate reduces FAD to FADH 2. Then electrons pass to Fe-S proteins which reduce Q to QH 2 Form 2 and 3 contains enzymes acyl-CoA dehydrogenase (oxidation of fatty acids) and glycerol phosphate dehydrogenase (oxidation of glycerol) which direct the transfer of electrons from acyl CoA to Fe-S proteins. Complex II does not contribute to proton gradient.

52. Ubiquinone Q: - lipid soluble molecule, - smallest and most hydrophobic of all the carriers - diffuses within the lipid bilayer - accepts electrons from I and II complexes and passes them to complex III. All electrons must pass through the ubiquinone (Q)- ubiquinole (QH 2 ) pair.

53. Complex III (ubiquinol-cytochrome c oxidoreductase) Transfers electrons from ubiquinol to cytochrome c. Consist of: cytochrome b, Fe-S clusters and cytochrome c 1. Cytochromes – electron transferring proteins containing a heme prosthetic group (Fe 2+  Fe 3+ ). Oxidation of one QH 2 is accompanied by the translocation of 4 H + across the inner mitochondrial membrane. Two H + are from the matrix, two from QH 2

54. Q-cytochrome c oxidoreductase is a dimer. Each monomer contains 11 subunits. Q-cytochrome c oxidoreductase contains three hemes: two b-type hemes within cytochrome b, and one c-type heme within cytochrome c 1. Enzyme also contains an iron-sulfur protein with an 2Fe-2S center.

55. Q cycle  two molecules of QH 2 are oxidized to form two molecules of Q,  one molecule of Q is reduced to QH 2,  two molecules of cytochrome c are reduced,  four protons are released on the cytoplasmic side,  two protons are removed from the mitochondrial matrix

56. Complex IV (cytochrome c oxidase) Transfers electrons from cytochrome c to O 2. Composed of: cytochromes a and a 3. Catalyzes a four-electron reduction of molecular oxygen (O 2 ) to water (H 2 O): O 2 + 4e - + 4H +  2H 2 O Translocates 2H + into the intermembrane space

57. Cytochrome c oxidase consists of 13 subunits and contains two hemes (two iron atom) and three copper ions, arranged as two copper centers.

58. The Catalytic Cycle of Cytochrome c Oxidise

59. The four protons used for the production of two molecules of water come from the matrix. The consumption of these four protons contributes to the proton gradient. Cytochrome c oxidase pumps four additional protons from the matrix to the cytoplasmic side of the membrane in the course of each reaction cycle (mechanism under study). Totally eight protons are removed from the matrix in one reaction cycle (4 electrons)

60. Cellular Defense Against Reactive Oxygen Species If oxygen accepts four electrons - two molecules of H 2 O are produced single electron - superoxide anion (O 2.- ) two electrons – peroxide (O 2 2- ). O 2.-, O 2 2- and, particularly, their reaction products are harmful to cell components - reactive oxygen species or ROS. DEFENSE superoxide dismutase (manganese-containing version in mitochondria and a copper-zinc-dependent in cytosol) O 2.- + O 2.- + 2H + = H 2 O 2 + O 2 catalase H 2 O 2 + H 2 O 2 = O 2 + 2 H 2 O antioxidant vitamins: vitamins E and C reduced glutathione

61. Proposed by Peter Mitchell in the 1960’s (Nobel Prize, 1978) Chemiosmotic theory: electron transport and ATP synthesis are coupled by a proton gradient across the inner mitochondrial membrane Mitchell’s postulates for chemiosmotic theory 1.Intact inner mitochondrial membrane is required 2.Electron transport through the ETC generates a proton gradient 3. ATP synthase catalyzes the phosphorylation of ADP in a reaction driven by movement of H + across the inner membrane into the matrix The Chemiosmotic Theory