1. Respiratory Chain The Oxidative Phosphorylation

2. Objectives ATP as energy currency Mitochondria and the electron transport chain organization Inhibitors of the electron transport chain Oxidative phosphorylation and the uncoupling proteins Inherited defects in oxidative phosphorylation. The role of mitochondria in apoptosis

3. Adenosine Triphosphate (ATP) Biochemists call ATP the ‘common currency of metabolism’ because it allows energy from fuel metabolism to be used for work, transport and biosynthesis. 1 gm fat gives 9 kcal while 1 gm carbohydrate and protein give 4 kcal only, about 40% of food energy is conserved as ATP, and the remaining 60% is liberated as heat.

4. Energy-rich molecules as glucose, fatty acids & amino acids are metabolized by a series of metabolic reactions yielding CO 2 and H 2 O & Energy is produced as ATP or heat

5. Electron Transport Chain (ETC) = Respiratory chain Electron transport chain (ETC) is the final common pathway in aerobic cells by which electrons and hydrogen (NADH & FADH2) derived from foodstuffs are transferred to oxygen to form water and finally produce ATP (energy) ETC is located in the inner mitochondrial membrane & is the final common pathway of metabolism (oxidative phosphorylation)

6. Electron Transport Chain (ETC) Energy-rich molecules as glucose or fatty acids are metabolized by a series of metabolic reactions yielding CO 2 and H 2 O. The metabolic intermediates of these reactions give electrons to specialized co-enzymes NAD and FAD to form NADH and FADH 2 which donate a pair of electrons to specialized set of electrons carriers, named the electron transport chain, ECT (Respiratory chain). As electrons are passed down the electron transport chain, they lose much of their energy. Part of this energy can be taken and stored by production of ATP from ADP and inorganic phosphate Pi (Oxidative phosphorylation). The remainder of the free energy not trapped as ATP is released as heat

7. Electron Transport Chain (ETC)

8. Organization of the respiratory chain: The inner mitochondrial membrane contains 5 separate enzyme complexes(Complexes I, II, III,IV and V ) a)Each complex accepts or donates electrons to relatively mobile carriers such as coenzyme Q and cytochrome C B) Each carrier of the electron transport chain can receive electrons from a donor and can subsequently donate electrons to the next carrier in the chain. The electrons finally combine with O 2 and proton (H + ) to form H 2 O. This requirement for O 2 makes the electron transport process named respiratory chain Electron transport chain

9. Components of respiratory chain: All components of the electron transport chains are protein in nature, except Coenzyme Q. 1)Complex I: contain enzyme called NADH dehydrogenase 2)Complex II : contain enzyme called flavoprotein dehydrogenase 3)Complex III: contains cytochrome b enzyme and cytochrome c 1 4) Complex IV: contains cytochrome a+a 3 (cytochromes are conjugated proteins with heme ring) Electron transport chain

10. 5)Complex V : ATP synthase it is formed of 2 subunits i)F1 subunit which protrudes into the matrix ii)F0 subunit which is present in the inner mitochondrial membrane The protons outside the inner mitochondrial membrane can re-enter the mitochondrial matrix by passing through F0-F1 complex.this results in synthesis of ATP from ADP+Pi Electron transport chain

11. Reactions of respiratory chain : 1)Entry via NADH+H : NADH+H can join the chain giving electrons to complex I to coenzyme Q to complex III to complex IV to final acceptor O 2 2)Entry via FADH 2 : FADH 2 obtained from complex II can join the chain directly giving electrons to coenzyme Q then complex III to complex IV to final acceptor O 2 The transport of a pair of electrons from NADH to oxygen via the ETC produces energy which is sufficient to produce 3 ATPs. The transport of a pair of electrons from FADH 2 to oxygen via the ETC produces sufficient energy to produce 2 ATPs. Electron transport chain

12. P : O ratios It is the ratio between numbers of ADP changed into ATP to the number of oxygen atom utilized by respiratory chain. It is 3:1 if electrons enter through NADH+H and it is 2:1 if electrons enter through FADH 2. Energy released from NADH & FADH2 entering ETC

13. Transfer of electrons across electron transport chain FREE ENERGY RELEASED Transport of protons (H + ) across the inner mitochondrial membrane from the matrix to the intermembrane space. This creates an electrical gradient with more +ve charge on the outside of the membrane than on the inside and a pH gradient with lower pH on outside. How the free energy generated by the transport of electrons by ECT is used to produce energy (ATP) Coupling of ECT to phosphorylation of ADP to ATP

14. Proton Pump Electron transport coupled to phosphorylation Electron transport is coupled to the phosphorylation of ADP by the transport of protons (H + ) across the inner mitochondrial membrane from the matrix to the intermembrane space. This creates an electrical gradient with more +ve charge on the outside of the membrane than on the inside and a pH gradient with lower pH on outside. the phosphorylation of ADP to ATP Protons reenter (goes back) the mitochondrial matrix by passing through a channel in the complex V (ATP synthase complex) giving an energy that are required for the phosphorylation of ADP to ATP.

15. Oxidative Phosphorylation (in mitochondria): Oxidation Oxidation: electron flow in electron transport chain (with production of energy) Phosphorylation Phosphorylation: phosphorylation of ADP to ATP 2- inhibitor of phosphorylation (Oligomycin)binds to ATP synthase closing the H+ channel preventing reentry of protons to the matrix & thus preventing phosphorylation of ADP to ATP 1- Inhibitors of oxidation via ETC are compounds that prevent the passage of electrons by binding to a component of the chain and subsequently blocking the oxidation/reduction reactions. As ETC and oxidative phosphorylation are tightly coupled, inhibition of the ECT also inhibits ATP synthesis. E.g: cyanide and CO poisoning

16. no Uncoupling proteins (UCPs) are located in the inner mitochondrial membrane leading to proton leak as they allow protons to reenter the mitochondrial matrix with no accompanying synthesis of ATP (no phosphorylation of ADP to ATP). No energy is utilized for the process of ATP synthesis, although ETC is functioning….i.e. The process of ETC is not coupled to posphorylation. However, energy is released in the form of heat. 3- Uncoupling proteins (UCP) The first discovered was uncoupling protein-1 (UCP1), formerly known as thermogenin, which is found exclusively in brown adipose tissue. Brown adipose tissue is abundant in the newborn and in some adult mammals, and it is brown because of its high content of mitochondria. In humans, brown adipose tissue is abundant in infants, but it gradually diminishes and is barely detectable in adults. UCP1 provides body heat during cold stress in the young and in some adult animals. It accomplishes this by uncoupling the proton gradient, thereby generating heat (thermogenesis) instead of ATP. Uncoupling proteins are expressed at high levels in hibernating animals, permitting them to maintain body temperature without movement or exercise.

17. Synthetic Uncouplers Synthetic uncouplers are compounds that can uncouple ETC & phosphorylation by increasing the permeability of the inner mitochondrial membrane to protons (thus will not reenter through ATP synthase) Examples: * 2,4-dintrophenol An uncoupler that causes electron transport to proceed at a rapid rate without phosphorylation & thus energy is released as heat rather than being used to synthesize ATP * High dose of aspirin (salicylates) uncouples oxidative phosphorylation causing fever (observed with toxic overdose of aspirin)

19. Inherited defects in oxidative phosphorylation Mitochondrial DNA (mtDNA) Mitochondrial DNA (mtDNA) is small circular DNA maternally inherited as mitochondria of sperm cell do not enter the fertilized ova. 13 polypeptide Mitochondrial DNA (mtDNA) codes only for 13 polypeptide (of total 120) required for oxidative phosphorylation. (while the remaining are synthesized in the cytosol & are transported into the mitochondria). alteration in mtDNA Defects of oxidative phosphorylation usually results from alteration in mtDNA (mutation rate 10 times more than that of nuclear DNA). Tissues with greater ATP requirement Tissues with greater ATP requirement (as CNS, sk.ms. & heart muscles, kidney & liver) are most affected by defects in oxidative phosphorylation.

20. pores allow cytochrome c to leave and enter the cytosol + proapoptotic factors, activates a family of proteolytic enzymes,the caspases cleavage of key proteins and resulting in the morphologic and biochemical changes characteristic of apoptotic cell death