Oxidative phosphorylation

It is like a summary of oxidative phosphorylation and reference to textbooks is required before presentation.

1. BY DEVI PRIYA SUGATHAN MSc BIOCHEMISTRY AND MOLECULAR BIOLOGY OXIDATIVE PHOSPHORYLATION

2. OXIDATIVE PHOSPHORYLATION Mitochondria are the site of oxidative phosphorylation in eukaryotes. It is the process in which ATP is formed as a result of transfer of electrons from NADH or FADH2 to O2 by a series of electron carriers. This transfer of electrons through the inner mitochondrial membrane leads to the pumping of protons out of the mitochondrial matrix. ATP is synthesized when protons flow back to the mitochondrial matrix through an enzyme complex.

3. Unlike substrate level phosphorylation, it does not involve phosphorylated intermediates. Thus, the oxidation of fuels and the phosphorylation of ADP are coupled by a proton gradient across the inner mitochondrial membrane. The actual synthesis of ATP is carried out by an enzyme called ATP synthase located in the inner mitochondrial membrane

4. Electron - Transfer Reactions in Mitochondria The discovery in 1948 by Eugene Kennedy and Albert Lehninger that mitochondria are the site of oxidative phosphorylation in eukaryotes marked the beginning of the modern phase of studies in biological energy transduction. Oxidative phosphorylation begins with the entry of electrons into the respiratory chain. Most of these electrons arise from the action of dehydrogenases that collect electrons from catabolic pathways and funnel into universal electron acceptors – nicotinamide nucleotides or flavin nucleotides. Kennedy and Lehninger

5. An overview of electron transport chain

6. Energy of electron transfer is efficiently conserved in a Proton Gradient The transfer of two electrons from NADH through the respiratory chain to molecular oxygen can be written as: NADH + H + + ½ O2 NAD + + H 2 O This net reaction is highly exergonic. For the redox pair NAD+/NADH, E’ is -0.320V and for the pair O 2 /H 2 O, E’ is 0.816V. The ∆E’ o for this reaction is therefore 1.14V and the standard free-energy change ∆G’ is -220kJ/mol (of NADH).

7. In actively respiring mitochondria, the actions of many dehydrogenases keep the actual [NADH]/[NAD] ratio well above unity, and the real free energy change for the reaction is substantially greater (more negative) than -220 kJ/mol. Much of this energy is used to pump protons out of the matrix. For each pair of electrons transferred to O2, four protons are pumped out by Complex I, four by Complex III and two by Complex IV.

8. CHEMIOSMOTIC MODEL Peter Mitchell proposed that electron transport and ATP synthesis are coupled by a proton gradient across the inner mitochondrial membrane. The transfer of electrons through the respiratory chain leads to the pumping of protons from the matrix to the cytoplasmic side of the inner mitochondrial membrane. The H+ concentration becomes lower in the matrix, and an electric field with the matrix side negative is generated. Peter Mitchell

9. Protons then flow back into the matrix to equalize the distribution. This flow of protons drives the synthesis of ATP by ATP synthase. The pH gradient and membrane potential constitute a proton-motive force that is used to drive ATP synthesis.

10. Proton Motive Force The energy rich unequal distribution of protons is called proton-motive force. The proton- motive force can be thought of as being composed of two components : a chemical gradient and a charge gradient. The chemical gradient for protons is the pH gradient and the charge gradient is created by the positive charge on the unequally distributed protons forming the chemical gradient.

11. ATP SYNTHESIS ATP synthase, a large enzyme complex of the inner mitochondrial membrane catalyzes the formation of ATP from ADP and Pi, accompanied by the flow of protons from the P to the N side of the membrane.

12. ATP SYNTHASE It is also called COMPLEX V. It is a large, complex enzyme that looks like a ball on a stick. Much of the “stick” part, called the F0 subunit, is embedded in the inner mitochondrial membrane. The 85-A diameter ball, called the F1 subunit, protrudes into the mitochondrial matrix. The F1 subunit contains the catalytic activity of the synthase.

13. F 1 Subunit It consists of five types of polypeptide chains ( α 3, β 3, γ , δ , ε ). The α and β subunits, which make up the bulk of the F1, are arranged alternately in the hexameric ring. Only the β subunit participate directly in catalysis. Below the α and β subunit is a central stalk consisting of the γ and ε proteins. The γ subunit includes a long helical coiled coil that extends into the center of the α 3, β 3 hexamer. The three β subunits is crucial for understanding the mechanism of ATP synthesis.

14. F Subunit It is a hydrophobic segment that spans the inner mitochondrial membrane. F0 contains the proton channel of the complex. This channel consists of a ring comprising from 10 to 14c subunits that are embedded in the membrane. A single a subunit binds to the outside of the ring.

15. The F0 and F1 subunits are connected in two ways: by the central γε stalk and by an exterior column. The exterior column consists of one a subunit, two b subunits and the δ subunit. We can think of the enzyme as consisting of a moving part and a stationary part : the moving unit or rotor consists of the c ring and the γε stalk and the stationary unit or stator is composed of the remainder of the molecule.

16. Role of proton gradient is not to form ATP but to release it from the synthase. The newly synthesized ATP does not leave the surface of the enzyme, it is the proton gradient that causes the enzyme to release the ATP formed on the surface. For the continued synthesis of ATP, the enzyme must cycle between a form that binds ATP very tightly and a form that releases ATP.

17. Binding – Change Mechanism Paul Boyer proposed the binding change mechanism for proton driven ATP synthesis. This proposal states that a β subunit can perform three sequential steps in the function of ATP synthesis by changing conformation. Interaction with the γ subunit make the three β subunits unequivalent. One β subunit can be in the L or loose conformation. This conformation bind ADP and Pi. Paul Boyer

18. A second subunit can be in the T or tight conformation. This conformation binds ATP with great avidity so that it will convert bound ADP and Pi into ATP. The final subunit will be in the O or open form where ATP is released and ADP and Pi binds to the O – form subunit. The rotation of the γ subunit drives the interconversion of these three forms and ATP is synthesized.

19. Proton flow around the c ring powers ATP synthesis The streaming of protons through the F0 “pore” causes the cylinder of c subunits and the attached γ subunit to rotate along its long axis which is perpendicular to the plane of the membrane. The γ comes in contact with the β subunit which forces the three β subunit to interact in such a way that one subunit assumes the β – empty conformation, its neighbor assumes β -ADP form and the other neighbor in β -ATP form. Thus one complete rotation of the γ subunit causes each β subunit to cycle through all three of its possible conformations and for each rotation three ATP are synthesized and released from the enzyme surface.

20. ADENINE NUCLEOTIDE TRANSLOCASE ATP and ADP do not diffuse freely across the inner mitochondrial membrane. A specific transport protein called Adenine nucleotide translocase (ATP- ADP Translocase) enables the ADP to enter into the mitochondrial matrix and ATP to cytosol. ATP 4- matrix + ADP 3- cytoplasm ADP 3- matrix + ATP 4- cytoplasm This antiporter moves four negative charges out for every three moved in, its activity is favored by the transmembrane electrochemical gradient, which gives the matrix a net negative charge.

21. PHOSPHATE TRANSLOCASE This transport process is also favored by the transmembrane proton gradient. This promotes symport of one H2PO4- and one H+ into the matrix. The process requires movement of one proton from the P to the N side of the inner membrane, consuming some of the energy of electron transfer.

22. ATP SYNTHASOME A complex of the ATP Synthase and both translocase is called the ATP Synthasome. This can be isolated from the mitochondria by gentle dissection with detergents, suggesting that the functions of these three proteins are very tightly regulated.

23. REGULATION OF OXIDATIVE PHOSPHORYLATION Oxidative phosphorylation is regulated by cellular energy needs. The intracellular [ADP] and the mass action ratio ( [ADP]/[ATP][Pi]) are measures of cells energy status. Normally this ratio is very high, so the ATP-ADP system is almost fully phosphorylated. When the rate of some energy requiring process increases, the rate of breaking down of ATP to ADP and Pi increases, lowering the mass-action ratio. When more ADP is available for oxidative phosphorylation, the rate of respiration increases, causing regeneration of ATP.

24. An inhibitory protein prevents ATP hydrolysis during hypoxia . When a cell is hypoxic, electron transfer to oxygen slows, and so does the pumping of protons. The proton motive force collapses and then under these conditions, the ATP synthase operates in reverse, hydrolyzing ATP to pump protons outwards and causing a drop in ATP. This is prevented by a small protein inhibitor, IF1, which simultaneously binds to two ATP Synthase molecules inhibiting their ATPase activity.

25. SUMMARY