1. How is the energy of Oxidation Preserved for the synthesis of ATP? ANS: Electron transfer to oxygen is accompanied by the formation of a high energy proton gradient. The Gradient arises by having protons pumped from the matrix side of the mitochondria to the inner membrane spaces Back flow of the protons to the matrix leads to the synthesis of ATP.

2. H+H+ H+H+ H+H+ H+H+ H+H+ H+H+ H+H+ H+H+ H+H+ H+H+ H+H+ H+H+ H+H+ H+H+ H+H+ H+H+ H+H+ H+H+ H+H+ H+H+ H+H+ H+H+ I II III IV NADH Cyt C O2O2 H2OH2O O2O2 H2OH2O O2O2 H2OH2O O2O2 H2OH2O O2O2 H2OH2O O2O2 H2OH2O O2O2 H2OH2O PROTON GRADIENT FORMATION (The Chemiosmotic Model of Energy Conservation Inner membrane space Matrix side

3. The Q-Cycle (Complex III-Cytbc 1 complex) Text p700 One electron goes on to Cytoc 1, the other stays in the Q cycle

4. QH 2 + 2 cytc 1 (oxidized) + 2H + N ( matrix side ) Q + 2 cytc 1 (reduced) + 4H + P ( inner membrane ) 2 protons come from QH 2 2 protons come from matrix

5. Free Energy Considerations (Chapt 11, p 398) 1. All substances in solution have a chemical potential 2. The chemical potential is related to concentration 3. Chemical potential is related to free energy 5. In terms of free energy of A: G A - G o’ A = RTln[A] 4. Given A. The chemical potential of A is said to be G A or the partial molar free energy of A. This equation says the free energy of A depends on the concentration of A This equation says the free energy of A depends on the concentration of A

6. A across a membrane 1. A difference in the concentration of A across a membrane creates a chemical potential difference 2. The difference is the difference of the chemical potential on either side:  G A = G A (in) - G A (out) = RT ln [A] out [A] in 3. A in to out: Equation assumes A goes from out to in Equation assumes A goes from out to in - RT ln [A] in [A] out = = RT ln [A] in [A] out

7. Proton Gradient Energy How much energy must be expended to transfer a proton from the matrix to the inner membrane? A A A A A A A A A outside inside A  G A = RT ln [A] in [A] out Since [A] in < [A] out  G A = negative Spontaneous A A A A  G A = RT ln [A] in [A] out  G A = positive Endergonic Requires ATP Since [A] in < [A] out = RT ln [A] out [A] in

8. Proton Gradient Energy A + – – – – – outside inside  G A = RT ln [A] in [A] out + + +  Z = charge on ion F = Faraday’s constant + ZF   G H+ = RT ln [H + ] out [H + ] in + ZF   G H+ = 2.3RT [pH (in) – pH (out) ] + ZF   G H+ = 2.3RT log [H + ] out -log[H + ] in + ZF  H+H+  G H+ = 2.3RT  pH+ ZF  The CHEMIOSMOTIC Principle Read Chapter 19 p703 Electrochemical potential If A is ionic (has a charge)

9. Problem: Calculate the pmf of a mitochondrial membrane that has a membrane potential of 168 mV and whose matrix pH is 0.75 units higher than its intramembrane space. = 4.12 kJ/mol + 16.21 kJ/mol pH gradient (20%)Membrane potential (80%) = 20.45 kJ/mol of protons  G A = 2.3RT  pH+ ZF  = 5.70 x (.75) + 96.5 x.168 = 5.70 kJ/mol x  pH + (1) 96.5 kJ/mol-volt x  volts The pH gradient and the Membrane potential both contribute to the proton motive force. The pH gradient and the Membrane potential both contribute to the proton motive force.

10. How is ATP made?  G = + 30.5 kJ/mol ADP + P i ATP + H 2 O F o F 1 ATPase Complex (ATP Synthase) 1. An ATP making machine 2. Driven by a proton gradient 3. Attached to the inner mitochondria membrane F 1 = stalk and lollypop F o = base

11. F1F1 FOFO F O F 1 ATPase (ATP Synthase) Matrix Intermembrane space H+H+ Binding-Change Model 3 non-equivalent sites

12. ADP + Pi ATP 3-Site Model of ATP Synthesis Loose Site (ADP and P i bind) Tight Site (ATP is formed and held) Open Site (ATP is released)  The flow of protons through F 1 makes the sites alternate much like a spinning propeller. F1F1

13. Older Model of ATP Synthesis NADH FMN CoQ Cyt b Cyt c1 Cyt c Cyt a+a3 O 2 ADP + PiATP Site 1 ADP + PiATP Site 2 ADP + PiATP Site 3 FADH 2 Model was tested by measuring P/O ratios

14. P/O Ratios P is phosphate taken up (incorporated into ATP) O is the oxygen taken up (measured as atomic oxygen) What is it? What is the significance? Compares substrate efficacy to form ATP Examples: P/O NADH~3 FADH 2 ~2 Succinate ~2 Assumed to be whole intergers based on the “coupling site” model of ATP synthesis (Equated to a pair of electrons traveling to O 2 )

15. Chemiosmotic Adjustment to P/O 10 protons are pumped for each electron pair from NADH 6 protons are pumped for each electron pair from FADH 2 4 protons are required to make one ATP 1 of the 4 is used in transport of ADP, Pi and ATP across mitochondrial membrane Therefore, 10/4 or 2.5 is the P/O ratio for NADH Therefore, 6/4 or 1.5 is the P/O ratio for FADH 2