ATP Synthesis via a Proton Gradient

1. ATP Synthesis via a Proton Gradient The two major 20th century biological discoveries: DNA structure and ATP synthesis

2. Evidence for the Chemiosmotic Hypothesis Electron transport and ATP synthesis are coupled by a proton gradient across the inner mitochondrial membrane.

3. ATP-Driven Rotation in ATP-Synthase: Direct Observation γ rotation with ATP present With low ATP 120-degree Incremental rotation Glass microscope slide

4. ATP Synthase with a Proton-Conducting (F0) and Catalytic (F1) Unit Intermembrane side F1 matrix unit contains 5 polypeptide chain types (α3, β3, γ, δ, ε) Proton flow from intermembrane space to matrix Matrix side

5. ATP-Synthase with Non-Equivalent Nucleotide Binding Sites F1 contains: α3, β3heximeric ring and γ, ε central stalk Central stalk and C-ring form the rotor and remaining molecule is the stator Top view Side view Matrix side

6. γ-Rotation Induces a Conformational Shift in the β Subunits Each β subunit interacts differently with the γ subunit ATP hydrolysis can rotate the γ subunit

7. Proton Flow Around C-Ring Powers ATP Synthesis Subunit C Asp protonation favors movement out of hydrophylic Subunit a to membrane region Deprotonation favors Subunit a movement back in contact with Subunit a

8. Proton Motion Across the Membrane Drives C-Ring Rotation

10. C-Ring Tightly Linked to γ and ε Subunits C-ring rotation causes the γ and ε subunits to turn inside the α3β3hexamer unit of F1 Columnar subunits (2 b) with δ prevent rotation of the α3β3hexamer unit What is the proton to ATP generation ratio?

11. Cytosolic NADH Movement to the Matrix via a Glycerol 3-Phosphate Shuttle NAD+ regeneration for glycolysis ATP synthesis capacity reduced Electron transport only What is DHAP converted into in glycolysis?

12. Alternative Q-Cycle Entry Points Complex I Complex II (citric acid cycle) Glycerol 3-phosphate shuttle Fatty acid oxidation (electron-transferring-flavoproteindehydrogenase)

13. Heart/Liver Malate-Aspartate Shuttle: Cytoplasmic NADH to Matrix

14. Malate-Aspartate Shuttle

15. Mitochondrial ATP-ADP Translocase Net movement down the concentration gradient for ATP (out of matrix) and ADP (into matrix) No energy cost

16. Mitochondrial Transporters for ATP Synthesis Net movement against the concentration gradient for Pi (into matrix) and charge balance -OH (out of matrix) Proton gradient energy cost

17. ATP Yield With Complete Glucose Oxidation

18. ADP Concentration Determines Oxidative Phosphorylation: Respiratory Control

19. Heat Generation by an Uncoupling Protein UCP-1 Brown adipose tissue rich in mitochondria for heat generation Pigs nest, shiver, and have large litters to compensate for lack of brown fat

20. ATP Synthesis Chemical Uncoupling What physiological effect might DNP have in humans?

21. Electron Transport Chain Inhibitors Toxins (e.g. fish and rodent poison rotenone) Site specific inhibition for biochemical studies What impact will rotenone have on respiration (O2 consumption)?

22. Proton Gradient Central to Biological Power Transmission

23. Chapter 21 Problems: 1- 3