1. Oxidative Phosphorylation: Structure and function of ATP synthase, mitochondrial transport systems, and inhibitors of Ox Phos Bioc 460 Spring 2008 - Lecture 30 (Miesfeld) Uncoupling proteins generate metabolic heat to protect vital organs during animal hibernation Dinitrophenol uncouples proton motive force and ATP synthesis The ATP synthase complex is the molecular motor of life

2. The ATP synthase complex is a molecular motor that undergoes protein conformational changes in response to proton motive force across the inner mitochondrial membrane. Mitochondrial shuttle systems are required to move metabolites across the impermeable inner mitochondrial membrane. Numerous inhibitors have been identified that interfere with ATP synthesis in mitochondria. The uncoupling protein UCP-1 converts redox energy into metabolic heat. Key Concepts in Oxidative Phosphorylation

3. The mitochondrial ATP synthase complex uses the proton-motive force generated via the electron transport system to synthesize ATP through protein conformational changes in a process called oxidative phosphorylation. In addition to generating ATP during aerobic respiration, a similar ATP synthase complex synthesizes ATP in response to proton motive generated by light-driven photosynthetic processes in plant chloroplasts.

4. Structure and Function of the ATP Synthase Complex Mitochondrial ATP synthase complex consists of two large structural components called F 1 which encodes the catalytic activity, and F 0 which functions as the proton channel crossing the inner mitochondrial membrane.

5. Three functional units of ATP Synthase 1.The rotor turns 120º for every H + that crosses the membrane using the molecular “carousel” called the c ring. 2.The catalytic head piece contains the enzyme active site in each of the three  subunits. 3.The stator consists of the α subunit imbedded in the membrane which contains two half channels for protons to enter and exit the F 0 component, and a stabilizing arm.

6. Proton movement through the ATP synthase complex forces conformational changes in the catalytic head piece in response to rotor rotation  top    bottom http://www.cnr.berkeley.edu/~hongwang/Project/ATP_synthase/MPEG_movies/F1_side_sp_2.mpeg

7. Proton movement through the ATP synthase complex forces conformational changes in the catalytic head piece in response to rotor rotation  top     http://www.cnr.berkeley.edu/~hongwang/Project/ATP_synthase/MPEG_movies/F1_top_sp_2.mpeg

8. Proton flow through F 0 alters the conformation of F 1 subunits Nucleotide binding studies revealed that it was the affinity of the  subunit for ATP, not the rate of ATP synthesis (or ATP hydrolysis in isolated F 1 fragments), that was altered by proton flow through the F 0 component. These studies showed that the dissociation constant (K d ) decreased by a million-fold in the presence of proton-motive force. Paul Boyer proposed the binding change mechanism of ATP synthesis to explain how conformational changes in β subunits control ATP production.

9. The binding change mechanism 1.The  subunit directly contacts all three  subunits, however, each of these interactions are distinct giving rise to three different β subunit conformations. 2.The ATP binding affinities of the three beta subunit conformations are defined as: T, tight; L, loose; and O, open. 3.As protons flow through F 0, the  subunit rotates such that with each 120º rotation, the β subunits sequentially undergo a conformational change from O --> L --> T --> O --> L --> etc. 4.The binding change mechanism model predicts that one full rotation of the  subunit should generate 3 ATP.

10. Looking down onto the catalytic head piece from the viewpoint of the mitochondrial matrix side. Follow the the conformational changes in the  1 subunit which will be O - L - T. 

11. From this viewpoint the  subunit rotates counter- clockwise. O L

12. ATP is formed in the  1 subunit but it is not released in the T state; release of ATP is the key step. L T Three more H+ pass through the c ring channel and the  subunit rotates another 120º.

13. ATP is released from the  1 subunit when it is in the O conformation. The  subunit sequence is O - L - T - O. T O

14. We will use 3 H + /ATP because it is a close approximation and it fits with the observation that 10 H + are translocated across the inner mitochondrial membrane for each NADH that is oxidized. The observed ATP currency exchange ratio of ~2.5 ATP/NADH is consistent with this because one full 360º rotation of the  subunit should produce 3 ATP for 9 H+ translocated. ~10 H+ translocated/NADH oxidized/~3ATP synthesized. The numbers don’t quite add up, but close enough

15. Boyer's model predicts that ATP hydrolysis by the F 1 headpiece should reverse the direction of the  subunit rotor. To test this idea, Masamitsu Yoshida and Kasuhiko Kinosita of Tokyo Institute of Technology used recombinant DNA methods to modify the , , and  subunits of the E. coli F 1 component in order to build a synthetic molecular motor.

16. Inter-membrane space side ATP hydrolysis Counter clockwise ATP synthesis Clockwise When they viewed the motor from the c ring side (inter-membrane space side), it was found to rotate counter clockwise for ATP hydrolysis. Normally for ATP synthesis, the  subunit rotates clockwise when viewed from the inter-membrane space.

17. Biochemical Application of the Oxidative Phosphorylation The F 1 component of the ATP synthase complex can be used as a "nanomotor" to drive ATP synthesis by attaching a magnetic bead to the  subunit and forcing clockwise rotation (viewed from the bottom) using electromagnets.

18. Clockwise, counterclockwise, matrix side, inter-mitochondrial membrane side - what is the take-home message? The structure-function relationships in the ATP synthase complex that catalyze ATP synthesis as a result of proton-motive force, are the same ones that catalyze ATP hydrolysis.

19. Energy released by ATP hydrolysis was the driving force for  rotation, not a proton gradient

20. The ATP synthase catalytic head piece rotates counterclockwise as viewed from the matrix side of the inner mitochondrial membrane during ATP synthesis. What direction does it rotate during ATP hydrolysis when viewed from the inter-membrane space? The opposite side of the membrane would be clockwise, but since it is also the opposite function (hydrolysis), the answer is counterclockwise. You didn’t have to know which direction it rotates a priori, I gave that information in the question. However, you did have to know that if you switch the orientation and/or the function, the rotation is reversed - this the key concept. Typical exam question on ATP motor rotation

21. In response to proton motive force, a H + will enter the half channel in the a subunit where it then comes in contact with a negatively charged aspartate residue in the nearby c subunit. How does H + movement through the c ring lead to  subunit rotation and subsequent conformational changes?

22. Transport Systems In The Mitochondria Key element of the Chemiosmotic Theory: The inner mitochondrial membrane must be impermeable to ions in order to establish the proton gradient. Biomolecules required for the electron transport system and oxidative phosphorylation must be transported, or "shuttled," back and forth across the inner mitochondrial membrane by specialized proteins For P i and ADP/ATP, this is accomplished by two translocase proteins located in the inner mitochondrial membrane.

23. Two Translocase Proteins 1.ATP/ADP Translocase –also called the adenine nucleotide translocase. –functions to export one ATP for every ADP that is imported. –an antiporter because it translocates molecules in opposite directions across the membrane. –for every ADP molecule that is imported from the cytosol, an ATP molecule is exported from the matrix. 2.Phosphate Translocase –translocates one P i and one H + into the matrix by an electroneutral import mechanism.

24. The Phosphate translocase functions as a channel The phosphate translocase functions as a symporter because both molecules are translocated in the same direction. This is an electroneutral translocation since the two charges cancel each other out.

25. Cytosolic NADH transfers electrons to the matrix via shuttle systems Numerous dehydrogenase reactions in the cytosol generate NADH, one of which is the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase. However, cytosolic NADH cannot cross the inner mitochondrial membrane, instead the cell uses an indirect mechanism that only transfers the electron pair (2 e - ), or two reducing equivalents, from the cytosol to the matrix using two different "shuttle" systems.

26. Most widely used shuttle is the malate-aspartate shuttle The key enzymes in this shuttle pathway are cytosolic malate dehydrogenase and mitochondrial malate dehydrogenase. Cytolosolic malate dehydrogenase Mitochondrial malate dehydrogenase This is the enzyme that replaces cytosolic NAD + during aerobic respiration.

27. The primary NADH shuttle in brain and muscle cells is the glycerol-3-phosphate shuttle The electron pair extracted from cytosolic NADH enters the electron transport chain at the point of Q rather than complex I.

28. The net yield of ATP from glucose oxidation in liver and muscle cells Let's add everything up to see how one mole of glucose can be used to generate 32 ATP in liver cells via the malate-aspartate shuttle, or 30 ATP in muscle cells which use the glycerol-3-phosphate shuttle.

29. The ETS and Ox Phos are functionally linked The role of the electrochemical proton gradient in linking substrate oxidation to ATP synthesis can be demonstrated by experiments using isolated mitochondria that are suspended in buffer containing O 2, but lacking ADP + P i and also lacking an oxidizable substrate such as succinate which has 2 e- to donate to the FAD in complex II of ETS.

30. Succinate increases rates of Ox Phos and O 2 consumption in isolated mitochondria, whereas, cyanide, CN -, which inhibits ETS, inhibits Ox Phos and O 2 consumption - what the...?

31. Dinitrophenol (DNP) dissipates the proton gradient by carrying H + across the inner mitochondrial membrane through simple diffussion-mediated transport The result is that carbohydrate and lipid stores are depleted in an attempt to make up for the low energy charge in cells resulting from decreased ATP synthesis; DNP short- circuits the proton circuit.

32. Dinitrophenol is a hydrophobic molecule that remains in the mitochondrial membrane as a chemical uncoupler for a long time - a very dangerous way to burn fat. J Anal Toxicol. 2006 Apr;30(3):219-22.

33. Oligomycin inhibits proton flow through the F o subunit of ATP synthase and blocks ATP synthesis, but oligomycin also blocks O 2 consumption - what the…? Addition of DNP to oligomycin-inhibited mitochondria leads to increased rates of O 2 consumption, but no change in rates of ATP synthesis - what the, what the, what the…?

34. Summary of known ETS and Ox Phos inhibitors

35. The UCP1 uncoupling protein, also called thermogenin, controls thermogenesis in newborn and hibernating animals