1. Oxidative phosphorylation pathway Lecture 15 Modified from internet resources, journals and books

2. Introduction Oxidation of NADH with phosphorylation of ADP to form ATP  processes supported by the mitochondrial electron transport assembly and ATP synthase (at inner mitochondrial membrane) electron transport assembly  comprised of a series of protein complexes that catalyze sequential oxidation reduction reactions

3. Principals of Reduction/Oxidation (Redox) Reactions Redox reactions  involve the transfer of electrons from one chemical species to another The oxidized plus the reduced form of each chemical species is referred to as an electrochemical half cell example of a coupled redox reaction is the oxidation of NADH by the electron transport chain: NADH + (1/2)O2 + H+ -----> NAD+ + H2O

4. Complexes of the Electron Transport Chain NADH is oxidized by a series of catalytic redox carriers that are integral proteins of the inner mitochondrial membrane free energy change in several of these steps is very exergonic Coupled to these oxidation reduction steps  is a transport process in which protons (H+) from the mitochondrial matrix are translocated to the space between the inner and outer mitochondrial membranes redistribution of protons  leads to formation of a proton gradient across the mitochondrial membrane

5. result of these reactions  redox energy of NADH is converted to the energy of the proton gradient This process  facilitated by a proton carrier in the inner mitochondrial membrane = ATP synthase  this carrier is coupled to ATP synthesis

7. Flow of electrons in the electron transport chain of oxidative phosphorylation The electrons pass through the proteins of complex I, four protons (H+) are pumped into the intramembrane space of the mitochondrion four protons are pumped into the intramembrane space as each electron pair flows through complexes III and as four electrons are used to reduce O2 to H2O in complex IV

8. continued all of the components of the pathway (exception NADH, succinate, and CoQ)  are integral proteins of the inner mitochondrial membrane the mitochondrial electron transport proteins are clustered into complexes: Complexes I, II, III, and IV Electrons flow through the complexes  Oxygen is the final electron acceptor, with water being the final product of oxygen reduction

9. Oxidative Phosphorylation The free energy available as a consequence of transferring 2 electrons from NADH or succinate to molecular oxygen is -57 and -36 kcal/mol Oxidative phosphorylation traps this energy as the high-energy phosphate of ATP In order for oxidative phosphorylation to proceed  two principal conditions must be met: the inner mitochondrial membrane must be physically intact so that protons can only reenter the mitochondrion by a process coupled to ATP synthesis

10. continued high concentration of protons must be developed on the outside of the inner membrane Electrons return to the mitochondrion through the integral membrane protein known as ATP synthase (or Complex V) In damaged mitochondria, permeable to protons  the ATP synthase reaction is active in the reverse direction acting as a very efficient ATP hydrolase or ATPase

11. Oxidative Phosphorylation For each pair of electrons originating from NADH  3 equivalents of ATP are synthesized proton gradient generated by electron transport contains sufficient energy to drive normal ATP synthesis

12. Regulation of Oxidative Phosphorylation flow of electrons through the electron transport system is regulated by the magnitude of the PMF (proton motive force) Under resting conditions, with a high cell energy charge  demand for new synthesis of ATP is limited energy demands are increased (vigorous muscle activity)  cytosolic ADP rises and is exchanged with intramitochondrial ATP while the rate of electron transport is dependent on the PMF, the magnitude of the PMF at any moment simply reflects the energy charge of the cell

13. Inhibitors of Oxidative Phosphorylation The pathway of electron flow through the electron transport assembly can be inhibited Some agents  inhibitors of electron transport at specific sites in the electron transport assembly Rotenone  Complex I Antimycin A  Complex III Cyanide  Complex IV Carbon Monoxide  Complex IV

14. Energy from Cytosolic NADH cytosolic NADH when oxidized via the electron transport system (in contrast to oxidation of mitochondrial NADH)  gives rise to 2 equivalents of ATP if it is oxidized by the glycerol phosphate shuttle and 3 ATPs if it proceeds via the malate aspartate shuttle glycerol phosphate shuttle  coupled to an inner mitochondrial membrane some tissues (heart, muscle)  mitochondrial glycerol-3- phosphate dehydrogenase is present in very low amounts  the malate aspartate shuttle is the dominant pathway for aerobic oxidation of cytosolic NADH the malate aspartate shuttle generates 3 equivalents of ATP for every cytosolic NADH oxidized (in contrast to the glycerol phosphate shuttle)

15. Brown Adipose Tissue and Heat Generation uncoupling of proton flow  releases the energy of the electrochemical proton gradient as heat this process  normal physiological function of brown adipose tissue Brown adipose tissue  gets its color from the high density of mitochondria in the individual adipose cells Newborn babies contain brown fat in their neck and upper back  serves the function of nonshivering thermogenesis muscle contractions that take place in the process of shivering  generates ATP, produces heat Nonshivering themogenesis  a hormonal stimulus for heat generation without the associate muscle contractions of shivering

16. continued The process of thermogenesis in brown fat  initiated by the release of free fatty acids from the triglycerides stored in the adipose cells The mitochondria in brown fat  contain a protein called thermogenein Thermogenein  acts as a channel in the inner mitochondrial membrane to control the permeability of the membrane to protons

17. continued norepinephrine is released in response to cold sensation  binds to -adrenergic receptors on the surface of brown adipocytes triggering the activation of adenylate cyclase Activated adenylate cyclase  leads to increased production of cAMP and the concomitant activation of cAMP-dependent protein kinase (PKA)  result being phosphorylation and activation of hormone-sensitive lipase released free fatty acids bind to thermogenin  triggering an uncoupling of the proton gradient and the release of the energy of the gradient as heat

19. Other Biological Oxidations Oxidase complexes (e.g. cytochrome oxidase)  transfer electrons directly from NADH and other substrates to oxygen, producing water Oxygenases  widely localized in membranes of the endoplasmic reticulum  catalyze the addition of molecular oxygen to organic molecules The chief components of oxygenase complexes  include cytochrome b5, cytochrome P450 and cytochrome P450 reductase There are many P450 isozymes; for example, up to 50 different P450 gene products can be found in liver, where the bulk of drug metabolism occurs

20. continued Enzymatic reactions involving molecular oxygen  usually produce water or organic oxygen in well regulated reactions having specific products under some metabolic conditions (e.g., reperfusion of anaerobic tissues)  unpaired electrons gain access to molecular oxygen in unregulated, non-enzymatic reactions The products = free radicals (quite toxic) free radicals, especially hydroxy radical  attack all cell components, including proteins, lipids and nucleic acids, potentially causing extensive cellular damage Tissues are replete with enzymes to protect against the random chemical reactions that these free radicals initiate

21. continued Superoxide dismutases (SODs) in animals  contain either zinc (Zn2+) and copper (Cu2+), known as CuZnSOD, or manganese (Mn2+) as in the case of the mitochondrial form SODs  convert superoxide to peroxide and thereby minimizes production of hydroxy radical (the most potent of the oxygen free radicals) Peroxides produced by SOD  also toxic --. detoxified by conversion to water via the enzyme peroxidase best known mammalian peroxidase is glutathione peroxidase, which contains the modified amino acid selenocysteine

22. continued Glutathione  important in maintaining the normal reduction potential of cells and provides the reducing equivalents for glutathione peroxidase to convert hydrogen peroxide to water In red blood cells  the lack of glutathione leads to extensive peroxide attack on the plasma membrane, producing fragile red blood cells that readily undergo hemolysis. Catalase (located in peroxisomes)  provides a reductant route for the degradation of hydrogen peroxide Mammalian catalase  has the highest turnover number of any documented enzyme