1. BIO 402/502 Advanced Cell & Developmental Biology I Section I: Dr. Berezney

2. Lectures 6 & 7 Electron Transport, Oxidative Phosphorylation & the ATP Synthase

3. ATP Synthesis Chemical energy produced in the cell is stored as ATP ATP synthesis occurs at the inner mitochondrial membrane of eukaryotes and the cell membrane in prokaryotes. In plants, ATP is synthesized along the thylakoid membranes Photosynthesis is a process by which plants convert light energy into ATP for glucose production. ATP synthase Electron transport chain Electron transport chain

4. Proton Gradient Across the Membrane: “Chemiosmosis” It is the universal mechanism of ATP production which involves the production of a proton motive force (pmf) based on a proton gradient across the membrane. Energy to establish this electrochemical proton gradient is provided by the energy released as electrons move to lower energy levels down the electron transport chain and the coupling of this free energy to the movement of protons across the IMM against the proton gradient [from matrix to IMS] ATP is synthesized by the ATP synthase F o F 1 complex : protons move with the proton gradient through F o F 1 to generate ATP [from IMS to matrix]

5. ATP Generation Glycolysis Conversion of glucose to pyruvate Net synthesis of 2 ATP by substrate level phosphorylation Krebs Cycle Converts pyruvate to acetyl CoA & carbon dioxide 10 molecules of coenzymes NADH and 2 of FADH 2 are produced. Results in synthesis of 30 ATP and 4 ATP molecules, respectively in the respiratory chain. Electron Transport (Respiratory) Chain The reduced coenzymes enter into the respiratory chain of the inner mitochondrial membrane Electron transport along the chain generates a proton electrochemical gradient and this is used to produce ATP

6. Electron Transport Chain Stepwise movement of electrons along the inner mitochondrial membrane respiratory chain proceeds from a lower to a higher (+) redox potential (E 0 ). Redox changes in mV are converted to free energy changes by the formula  G 0 = -nF  E 0 ; F=23.063 (Kcal/Vmol ****[ΔE o = E o(red) - E o(ox) ] -  G corresponds to release of free energy and hence more negative values represent higher energy levels of the transporting electrons H + in  [matrix] H + out  [IMS] III III IV

7. Electron Transport Chain contd… -------------------------------------------------------------------------------- ------------- The Four Electron Transport Complexes in the Inner Mitochondrial Membrane Respiratory Chain -------------------------------------------------------------------------------- -------------- (a) Complex I NADH-CoQ reductase ………  G = - 16.6 kcal/mol ( ATP ) (b) Complex II Succinate CoQ reductase ….  G = - 1.6 kcal/mol ( no ATP ) (c) Complex III CoQ- Cytochrome c reductase,  G = - 10.1 kcal/mol ( ATP ) (d) Complex IV Cytochrome c oxidase…………  G = -19.8 kcal/mol ( ATP )

8. Glucose  10 NADH and 2 FADH 2 ; NADH  3 ATP; FADH2  2 ATP;Total ATP = 34 The Four Complexes of the Respiratory Chain Complex I 45 subunits ~1000 kDa Complex III 11 subunits ~240 kDa Complex II 4 subunits ~125 kDa Complex IV 13 subunits ~ 200 kDa

9. Free energy released during electron transport in the RC is stored as a proton generated electrochemical gradient across the membrane which is composed of two components: (a) An electric potential (  G e = zF  E m ) (b) Proton chemical gradient (  G c = (2.3) RT log [H + ] I = - (2.3) RT  pH ) Calculation of ∆G for electrochemical gradient and the pmf [H + ] o (c) If  pH = 1, and ΔE m = - 160 mV:  G t =  G e +  G c = zF  E m - (2.3) RT  pH = - 3.7+ - 1.4 = - 5.1 kcal/mol for 1 proton (mol) to move down the electrochemical gradient Proton motive force (pmf) is the proton electrochemical gradient across the membrane expressed in volts or mV [Remember: ΔGo= zFΔEo]. (a)Proton motive force =  - 2.3 RT  pH =  - 59  pH ;  is the membrane electrical potential. (b) Assume respiring mitochondria have a  of -160 mV and  pH is 1. Thus pmf or Δp = -160 mV - (59) (1) = - 160 – (59) = - 219 mV. (c) ΔG = zF ΔE = 23.06 ( - 0.219 V) = - 5.1 kcal/mol F

10. Proton Motive Q Cycle CoQ binds 2 protons on matrix side One electron is transported via FeS protein and cyt c 1 to cyt c The other electron goes through cyt b’s to CoQ. This CoQ is fully reduced by repeating this step. # protons transported by 100 CoQ molecules is: 200+100+50+25+12.5 + … n = 400

11. Cytochrome C Oxidase (complex IV) Transport

12. Structure of the Cytochrome C Oxidase Monomer The heme groups are shown in blue and red and copper sites in green The catalytic core consists of I yellow, II blue, III pink The entire complex consists of 13 subunits

13. Structure of Beef Heart Cytochrome Oxidase The protein is a dimer of two 13 monomers 3 dimensional structure of beef heart cytochrome oxidase at 2.8 angstrom resolution

14. ATP Synthase: An Electrical Mechanochemical Molecular Complex

15. On the the Inner Mitochondrial Membrane (IMM), the F 0 F 1 Complex or ATP Synthase Uses the Proton Gradient Generated by Electron Transport of the Respiratory Chain to Synthesize ATP Electron transport chain

16. Change in pH indicates that for each electron pair transported from NADH to oxygen, 10 protons are transported out of the matrix. Correlating with the return of pH to normal is the synthesis of ATP by the mitochondria. Proton transport is abolished by addition of mild detergent that makes the IMM permeable. Evidence that electron transport in mitochondria is coupled to proton translocation

17. Demonstrating Function for Mitochondria F 1 Particles This “reconstitution experiment” demonstrates that F 1 are required for ATP synthesis and not for electron transport in the mitochondrial vesicles F1F1

18. The measured pH indicates that two protons are translocated per reduced oxygen atom. No ATP synthesis in presence of ADP and P i. Not Shown: If F o F 1 complexes are inserted into one of the RC complexes, then ATP synthesis can be measured in correlation with electron transport This is evidence that F o F 1 is the ATP synthase Liposome Experiment Demonstrating Coupling of Electron Transport by Cytochrome Oxidase to Proton Translocation and F o F 1 as the ATP synthase

19. ATP Synthesis in Thylakoid Membranes An artificially imposed pH gradient across the chloroplast thylakoid membrane can drive ATP synthesis in the absence of electron transport !!!!! The Famous Jagendorf Experiment

20. 3 D Model of ATP Synthase: An Electrical Mechano- Chemical Molecular Complex The F o portion is composed of integral transmembranous proteins a, b and 9-14 copies of c which forms a ring-like structure in the plane of the membrane. The F 1 head piece is composed of a hexagonal array of alternating  and  subunits, a central  protein with a helical coil that associates with  and  proteins and extends into the c protein ring in the F o.

21. Atomic Force Microscopy of C-subunit Ring Structures Isolated from Chloroplast ATP Synthase and Inserted Into Liposomes

22. Synthesis of ATP: Rotary Catalysis ATP is synthesized by coupling the energy liberated during proton translocation through the F o F 1 to a motive force that rotates the C ring structure and the attached  subunit.  -subunits contain the catalytic sites of ATP synthesis. 120 degree units of rotation of the  protein around the stationary  /  hexagonal array results in altered associations of the  protein with the  protein forming the L, T and O states for the 3 β-subunits. ATP is produced in the T state where the ∆G = ~ 0. Each rotation of 360 degrees of the γ subunit results in 3 ATP, one for each β-subunit. The model shows the rotation as arbitrarily clockwise. ∆G = ~ 0

23. Direct Visualization of Rotary Catalysis From these results it was determined that the gamma protein rotates like the camshaft in a rotary motor at a maximum rate of ~8,000 rpm! Magnetic bead experiments shows rotation in the opposite direction (clockwise) for ATP production! Add ATP F 1 complex F 0 F 1 complex

24. A revolving magnetic field to rotate clockwise a magnetic bead attached to the gamma subunit of a single F1 complex results in ATP synthesis. Revolving electromagnetic field Clockwise rotation led to the synthesis of 3 ATP’s for every 360 o turn. When the magnetic field was switched off, the gamma subunit revolved in the reverse direction, driven by the recently synthesized ATP. F 1 complex

25. Speculative Model for Coupling of Proton Transport to the Rotation of the c Ring of F o Proton binding to Asp61 of c- subunit [on IMS side] induces a conformational change in the c subunit that causes the ring to move by 30-40 degrees. Each subsequent c picks up a proton. Bound protons are carried in full circle rotating 30-40 degrees at a time and are then released into the matrix compartment and the c subunit is free to bind another proton. For one rotation of c-ring: 12 protons (30°); 9 protons (40°) Site directed mutagenesis of Asp61 prevents proton tranlocation across F 0.