Lecture 6 BCHM2971

1. Lecture 6 BCHM2971 Biochemical thermodynamics: ATP and redox reactions. Oxygen’s double edged sword

2. Thermodynamics and mechanisms of storing and spending energy Proton gradient fuel ADP spend WORK release store store spend NAD NADH C02 ATP Glycolysis Krebs e- transport chain Redox and E Oxidative phosphorylation Free energy DG coupling

3. Plan for today’s lecture • Free-energy currency is "spent" to drive nonspontaneous reactions • DG and coupling • Why is ATP the currency of free-energy? • Redox cycles of e- and H+ transfer: • redox potentials (DE ) • Mechanism of e- and H+ transfer: • Complex 4 of the electron transfer chain • Oxygen as the final acceptor of electrons

4. Why eat? • most metabolic reactions are not spontaneous • require a source of free energy = DG • Energy released from food is eventually ‘saved’ in ATP  ‘spent’ to drive energetically unfavourable reactions

5. Free energy change (DG) • Free energy change (DG) of a reaction determines its spontaneity • negative DG spontaneous ( products) ie: G products < G reactants R = gas constant; T = temp.

6. standard free energy change pH 7 ([H+] = 10-7M) reactants & products =1M free energy change of reactionunder ‘other’ conditions (eg in the cell) DG Value depends on actual [products] and [reactants]

7. Hydrolysis of ATP • useful free-energy ‘currency’ • dephosphorylation reaction is very spontaneous ATP  ADP + Pi (DGo' = -31 kJ/mol)DG<0

8. Spontaneous? • Spontaneous does not indicate how quickly a reaction occurs • ATP (and pals) are kinetically stable (usually have free energies of activation) • Rate low without enzyme Activation energy energy -ve DG reaction

9. Spontaneous? Why doesn’t ATP explode?? • Spontaneous does not indicate how quickly a reaction occurs • ATP (and pals) are kinetically stable (usually have free energies of activation) • Rate low without enzyme Activation energy (lowered by enzyme) energy -ve DG reaction

10. Spontaneous? • Kinetic stability essential: • reaction energy is then  Controllable by catalysis  Can be coupled to useful reactions Activation energy (lowered by enzyme) energy -ve DG reaction

11. What makes the bonds in ATP‘high-energy”? • Phosphoanhydride bonds tend to have a large negative DG (-30.5 kJ.mol-1) • NB: bond energy is not necessarily high, just the free energy of hydrolysis. Phosphoester bond Phosphoanhydride bonds Adenine gP bP aP O O CH2 Ribose ATP

12. Two strongly e- withdrawing groups compete for p e- of the bridging oxygen No such competition in the hydrolysis product more stable 1. PhAnH bond has less stable resonance than its product hydrolysis

13. At pH 7, ATP has 3 –ve charges Repulsion is relieved by hydrolysis  more stable 2. PhAnH bond has greater electrostatic repulsion than its product hydrolysis

14. 3. Solvation energy • Phosphoanhydride bond has smaller solvation energy than product  favours hydrolysis

15. Measure of tendency of compound to transfer ~P to H20 ATP is intermediate! Can accept ~P from compounds above Or donate ~P to compounds below Phosphoryl group-transfer potential

16. Other high energy compounds Other phosphorylated compounds Phosphocreatine Thioesters CoenzymeA (you will meet this in other lectures)

17. Phosphocreatine • Higher P-group transfer potential than ATP • ‘reservoir’ of ~P for rapid ATP regeneration Maintains constant level of ATP by swapping ~P =reversible ‘substrate-level phosphorylation’ in tissues with high need (muscle, nerve) When ATP is low, phosphocreatine can lend a P to ADP to make ATP. When ATP is replenished by catabolism, P is ‘paid back”. When  ATP  P phosphocreatine creatine P ADP ATP When  ATP

18. Why create high energy compounds? • spontaneousreactions DG<0 are often coupled with non-spontaneous reactions (DG>0) to drive them forward. • The free-energy change (DG) for coupled reactions is the sum of the free-energy changes for the individual reactions. DGcoupled = DG reaction 1 + DG reaction 2

19. Thus, ATP  ADP +Pi(DG<0) is coupled with non-spontaneous reactions (DG>0) to drive them forward. Glucose glucose-6-P + H20 DG = 13.8 kJ.mol-1 hexokinase DG = -30.5 kJ.mol-1 ATP +H20 ADP +Pi DG = -16.3 kJ.mol-1 Glucose + ATP Overall: spontaneous! glucose-6-P + ADP

20. eg oxidative phosphorylation Spontaneous H+ movement against gradient coupled to ATP synthesis Energy coupling with ion gradientEnergy can also be stored as an ion gradient ADP Proton gradient -ve DG +ve DG ATP

21. How does energy from food get transferred to ATP for storage? Controlled cycles of oxidation and reduction

22. Electron transport chain (ETC) e- H Sequential transfer of H: (2e- and H) from fuels indirectly provides free energy for production of ATP. What causes transfer of e- and H+? How does this release energy to create an ion gradient?? Remember redox potentials? CO2 glucose OXIDATION REDUCTION e- e- NAD+ NADH e- e- O2 H2O OXIDATION REDUCTION I H Cyt C III IV Q e- e-

23. e- B reduced Aoxidised A reduced OXIDATION REDUCTION B oxidised gain electrons, gain H lose O The tendency of a substance to undergo reduction = E°’ (reduction potential) E°’= Affinity for electrons DE °' =E °‘ (acceptor) – E °‘ (donor)

24. Reduction Potential and Relationship to Free Energy DE °' =E °'(acceptor) – E °'(donor) DGo' = – nFDE °' **Don’t learn these equations! Just understand the implications of +ve or –ve values # electrons transferred Faraday constant

25. DGo' = – nFDE °' • An electron transfer reaction is spontaneous(-veDG) if DE°‘ is+ve ie: when E °' of the acceptor >E °' of the donor Electrons spontaneously flow from low  high reduction potentials

26. e- B reduced Spontaneous if... Aoxidised A reduced OXIDATION REDUCTION B oxidised acceptor has higher DE

27. NAD accepts e- and H+ from fuel NADH NADH donates e- and H+ to ETC thermodynamics of the ETChain Oxidised reduced Hydride ion = 2e + H+ Accepts e- from fuel In ETC

28. NADH oxidation is spontaneous and releases free energy E°’=-0.3 V NAD++ H++ 2e- NADH oxidised reduced E°’ = +0.8 V H2O ½ O2+ 2H++ 2e- DE °' =E °'(acceptor) – E °'(donor)DE °‘ = 0.8 – (-0.3) = 1.13V O2 has greatest affinity for e-NADH becomes the e- donor

29. NADH oxidation is spontaneous and releases free energy NAD++ H++ 2e- NADH OXIDATION oxidised reduced REDUCTION H2O ½ O2+ 2H++ 2e- DE °‘= 1.13VDGo' = – nFDE °‘ - ve +ve

30. electrons are not transferred directly from NADH to O2 • rather pass through a series of intermediate electron carriers • Why? This allows energy released to be coupled to protons pump. • ultimately responsible for coupling the energy of redox to ATP synthesis.

31. Electrons spontaneously flow from low to high reduction potentials Increasing E

32. One example in more detail: Complex IV (cytochrome c oxidase) Transmembrane spanning a-helices

33. Complex IV (cytochrome c oxidase) • Catalyses final reduction in the ETC • O2 + 4 H+ + 4 e- 2 H2O (irreversible) • The four electrons are transferred into the complex one at a time from cytochrome c. • Results in pumping of 4 H+ across the membrane.

34. haem a3, (Fe) CuB Has 4 metal ‘redox centers’ • CuA(=2 Cu atoms) • haem a (Fe) Ions in close proximity = binuclear complex

35. e- are passed one at a time FIRST: 2e- passed from cytC by haem a-CuA to binuclear center e- Cyt C

36. H+ from matrix and hydroxyl from binuclear center  H2O 2e- were passed from cytC by haem a-CuA to fully reduce Fe and Cu in the binuclear center O O O- H H H So far… Fully reduced Fully oxidised e- H+ e- e- e- Fe3+Cu2+ Fe2+ Cu+ Tyr Tyr O H H

37. e- e- Fe2+ Cu+ Tyr O O O- O H H H H Then, O2 binds Fully reduced O e- H+ O e- O e- e- Fe3+Cu2+ Fe2+ Cu+ O Tyr Tyr O H H This O2 is going to become O22-It’s going to need 4 e-

38. 4e- are rearranged Only 3e- can be donated by the metal ions (see why?) So 1e- ALSO must be donated temporarily from tyrosine  OXYFERRYL complex e- e- Fe2+ Cu+ Tyr O O O- O H H H H The tricky bit!! O Fully oxidised e- H+ O e- O e- e- Fe3+Cu2+ Fe2+ Cu+ O Tyr Tyr O H H Fe2+ - 2e-  Fe4+Cu + - 1e-  Cu2+ Tyr-OH -1e- -H+  Tyr-O. H e- e- O- e- O2- Fe4+Cu2+ e- Tyr O

39. O22- shared between Cu and Fe

40. 1 more e- passed in via haem3-CuA to binuclear complex  Reconverts tyrosine e- e- Fe2+ Cu+ Tyr O O O- O O H H H H H O Fully oxidised e- H+ O e- O e- e- Fe3+Cu2+ Fe2+ Cu+ O Tyr Tyr O H H O H H H e- e- e- O- e- O2- e- O2- Fe4+ Cu2+ Fe4+ Cu2+ e- e- Tyr Tyr O H e- And more H+ H2O H

41. And one more H+ e- e- Fe2+ Cu+ Tyr O O O- O O H H H H H 4th e- passed via h3CuA Regenerates Fe3+: Completed cycle! O Fully oxidised e- H+ O e- O e- e- Fe3+Cu2+ Fe2+ Cu+ O Tyr Tyr O H H e- H O H H H e- e- e- O- e- O2- e- O2- Fe4+ Cu2+ Fe4+ Cu2+ e- e- Tyr Tyr O e- H H

42. H+ H+ H+ H+ Meanwhile pumps 4 H+ were pumped to proton gradient H+ H+ H+ O H+ O

43. O2 as final e- acceptor • Strong e- acceptor (high E) • Provides  thermodynamic force • Also, controllable: reacts slowly unless catalysed by enzyme

44. Disadvantages • O2 + 4 e- safe 2H20 • BUT partial reduction  DANGER!!! • O2 + e-  O2– (superoxide) • Can extract e- from other molecules  ‘free radicals’ • Oxidisation of membranes, DNA, enzymes • Implicated in Alzheimers, Parkinsons, aging

45. Summary • Hydrolysis of ATP is spontaneous (–ve DG) • Free energy of ATP coupled to non-spontaneous reactions • Phospho-anhydride bond is ‘high energy’ • Electrons spontaneously flow from low to high E Food NAD e- transfer chain O2 • Free energy used to create proton gradient that is then ‘spent’ to make ATP

46. Do NOT learn these values! Just know which are +ve or –ve/ spontaneous or not…understand concept of coupling!! The individual reactions are: • oxidation NADH  NAD+ + H+ +  2e-DGo= -158.2 kJspontaneous • reduction   ½ O2 + 2H+ + 2e- H2O DGo= -61.9 kJspontaneous • phosphorylation ADP   ATP DGo= +30.5 kJnonspontaneous • The net reaction is obtained by summing the coupled reactions, ADP + NADH + ½ O2 + 2H+       ATP + NAD+ + 2 H2O DGo= -189.6 kJspontaneous Coupled non-spontaneous work