L4.2.pdf

SESSION-4, LCETURE-1 OXIDATIVE PHOSPHORYLATION Dr. Aveen Hassan Mustafa MBChB, MSc, FKBMS-Chemical Pathologist [email protected] LEARNING OUTCOMES 1. Describe the key features of oxidative phosphorylation. 2. Explain the processes of electron transport & ATP synthesis & how they are coupled....

SESSION-4, LCETURE-1 OXIDATIVE PHOSPHORYLATION Dr. Aveen Hassan Mustafa MBChB, MSc, FKBMS-Chemical Pathologist [email protected] LEARNING OUTCOMES 1. Describe the key features of oxidative phosphorylation. 2. Explain the processes of electron transport & ATP synthesis & how they are coupled. 3. Describe how, when and why uncoupling of these processes occur in some tissues. 4. Compare the processes of oxidative phosphorylation & substrate level phosphorylation. 2 3 OXIDATIVE PHOSPHORYLATION LO-1 § Is a process in which the free energy of oxidation of NADH and FAD2H is used by the electron transport system to pump protons into the intermembrane space. § The energy produced when these protons reenter the mitochondrial matrix is used to synthesize ATP. LO-1 KEY FEATURES OF OXIDATIVE PHOSPHORYLATION § Oxidative § The Phosphorylation is Stage 4 of catabolism. complete oxidation of glucose: C6H12O6 + 6O2 à 6 H2O ΔGo = -2,870 kJ/mole By the end of stage 3 (TCA cycle): § All C-C bonds have been broken, and C-atoms oxidised to CO2 § All C-H bonds have been broken, and H-atoms (H+ and e-) transferred to NAD+ and FAD. All of the energy from the breaking of these bonds has gone to: § ATP/GTP formation (2 in glycolysis, 2 in the TCA cycle) § Chemical bond energy of the e- in NADH/FADH2 5 KEY FEATURES OF OXIDATIVE PHOSPHORYLATION § LO-1 NADH and FAD2H contain high energy electrons that can be transferred to oxygen through a series of carrier molecules, releasing large amounts of free energy. 6 KEY FEATURES OF OXIDATIVE PHOSPHORYLATION LO-1 § This energy can be used to drive ATP synthesis in the final stage of catabolism (oxidative phosphorylation), occurring in the inner mitochondrial membrane. § Two processes are involved: Ø Electron Transport, electrons in NADH and FAD2H are transferred through a series of carrier molecules to oxygen, releasing free energy. Ø ATP synthesis, the free energy released in electron transport drives ATP synthesis from ADP + Pi 7 ELECTRON TRANSPORT LO-2 o Carrier molecules transferring electrons to molecular oxygen are organized into a series of four highly specialized protein complexes spanning the inner mitochondrial membrane. o Electrons are transferred from NADH (and FAD2H) sequentially through the series of complexes to molecular oxygen with the release of free energy. o Three of the complexes, in addition to transferring electrons, also act as proton translocation complexes (proton pump). 8 ELECTRON TRANSPORT § Complex I (NADH-Q oxidoreductase): oxidizes NADH and transfers electrons to coenzyme Q § Coenzyme Q (Ubiquinone) § Complex II (Succinate-Q reductase): oxidizes FADH2 and transfers electrons to coenzyme Q § Complex III (Q-cytochrome c oxidoreductase): passes the electrons to cytochrome c § Complex IV (Cytochrome c oxidase): passes the electrons to O2 9 ELECTRON TRANSPORT LO-2 10 PROTON MOTIVE FORCE (PMF) LO-2 o Free energy from electron transport is used to move protons from the inside to the outside of the inner mitochondrial membrane via proton translocating complexes. o The membrane itself is impermeable to protons and as electron transport continues the concentration of protons outside the inner membrane increases. o The proton translocating complexes therefore transform the chemical bond energy of the electrons into an electro-chemical gradient. o This is known as the Proton Motive Force. o NADH has more energy than FAD2H and so uses all three proton translocating complexes while FAD2H only uses two. o This process requires oxygen, as it is the last electron acceptor. 11 ATP SYNTHESIS o LO-2 ATP Hydrolysis results in the release of energy (ΔGo = -31kJ/mol). ATP + H2O à ADP + Pi ΔGo = -31 kJ/mole o Therefore for the synthesis of ATP from ADP and Pi, + 31 kJ/mol of energy is required to drive the reaction. o This energy is derived from the (pmf) that has been produced across the inner mitochondrial membrane by electron transport. o Protons can normally only re-enter the mitochondrial matrix via the ATP synthase complex, driving the synthesis of ATP from ADP and Pi. § The greater the PMF the more ATP synthesized. § The oxidation of 2 moles of NADH gives 5 moles of ATP § The oxidation of 2 moles of FAD2H gives 3 moles of ATP 12 12 COUPLING OF ELECTRON TRANSPORT AND ATP SYNTHESIS § ET LO-2 and ATP Synthesis are tightly coupled. One does not occur without the other. § The mitochondrial concentration of ATP plays an important role in regulating both processes. § When ATP concentration is high: • The ADP concentration is low and the ATP synthase stops (lack of substrate) • This prevents H+ transport back into the mitochondria • The H+ concentration outside increases to a level that prevents more protons being pumped to the outside • In the absence of proton pumping, electron transport stops • The reverse occurs when [ATP] is low. 14 UNCOUPLERS LO-3 Some substances (eg dinitrophenol, dinitrocresol) increase the permeability of the inner mitochondrial membrane to protons. o Therefore protons being pumped out by electron transport can reenter without passing through the ATP synthase complex. o The two processes become uncoupled so the p.m.f. is dissipated as heat. o Proton leak is physiologically important and accounts for 20-25% of the basal metabolic rate (BMR). o 15 UNCOUPLING PROTEINS (UCPS) LO-3 § The function of UCPs is to uncouple ET from ATP production to produce heat. § The proteins are located in the inner mitochondrial membrane and allow a leak of protons across the membrane. § UCP1 - (previously known as thermogenin) is expressed in brown adipose tissue and involved in non-shivering thermogenesis enabling mammals to survive the cold. § UCP2 – Quite widely distributed in the body. Research suggest it is linked to diabetes, obesity, metabolic syndrome and heart failure. § UCP3 – Found in skeletal muscle, brown adipose tissue and the heart. It appears to be involved in modifying fatty acid metabolism and in protecting against ROS damage. 16 UNCOUPLING PROTEINS (UCPS) LO-3 § Noradrenaline – Is released from the sympathetic nervous system and stimulates lipolysis releasing fatty acids to provide fuel for oxidation in brown adipose tissue. § NADH and FAD2H are formed as a result of β-oxidation of the fatty acids. § NADH and FAD2H drive ET and increase p.m.f. § However, noradrenaline also activates UCP1, allowing protons to cross the inner mitochondrial membrane without passing through the ATP synthase complex. § The higher p.m.f. is dissipated as heat. 17 INHIBITORS OF ELECTRON TRANSPORT LO-3 § ET is inhibited under anaerobic conditions and by a number of substances including carbon monoxide and various poisons (cyanide, rotenone, antimycin). § Under these conditions NADH & FAD2H cannot be oxidized by electron transport. § As a consequence there is no energy to drive the pumping of protons and a p.m.f cannot be created. § Without the p.m.f. ATP cannot be synthesised and no heat is generated. § Irreversible cell damage rapidly occurs. 18 OXIDATIVE PHOSPHORYLATION VS SUBSTRATE LEVEL PHOSPHORYLATION LO-4 Oxidative Phosphorylation Substrate Level Phosphorylation Requires membrane associated complexes (inner mitochondrial membrane) Requires soluble enzymes. (Cytoplasmic and mitochondrial matrix) Energy coupling occurs indirectly through generation and subsequent utilisation of a proton gradient (p.m.f.) Energy coupling occurs directly through formation of a high energy of hydrolysis bond (phosphoryl-group transfer) Cannot occur in the absence of oxygen Can occur to a limited extent in absence of oxygen Major process for ATP synthesis in cells that require large amounts of energy Minor process for ATP synthesis in cells that require large amounts of energy 19