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
Keep this diagram in mind during the lecture. The theme is how free energy is released from food, captured and stored first in the reducing power of NADH, then the proton gradient, then in high energy bonds of ATP and finally ‘spent’ to do work (thermodynamically unfavourable, non-spontaneous reactions of life)
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? require a source of free energy = DG
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
Don’t learn this equation…if needed, it will be provided in the question.
R = gas constant; T = temp.

6. Value depends on actual [products] and [reactants]
standard free energy change
pH 7 ([H+] = 10-7M)
reactants & products = 1M
free energy change of reaction under ‘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
This should be revision from MBLG1001. The old lecture will be on the weeb if you need to revise it (just skip to that part of the lecture)
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. 1. PhAnH bond has less stable resonance than its product
Two strongly e- withdrawing groups compete for p e- of the bridging oxygen
No such competition in the hydrolysis product more stable
hydrolysis

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

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

15. Phosphoryl group-transfer potential
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

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

17. Phosphocreatine When  ATP  When  ATP
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?
spontaneous reactions 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 = kJ.mol-1
ATP +H20
ADP +Pi
DG = kJ.mol-1
Glucose + ATP
Overall: spontaneous!
glucose-6-P + ADP

20. Energy coupling with ion gradient Energy can also be stored as an ion gradient
eg oxidative phosphorylation
Spontaneous H+ movement against gradient coupled to ATP synthesis
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)
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?
glucose
CO2
OXIDATION
REDUCTION e- e-
NAD+
NADH e- e- O2
H2O
OXIDATION
REDUCTION I H
Cyt C
III IV Q e- e-
Electron transport chain (ETC)

23. = E°’ (reduction potential) DE °' = E °‘ (acceptor) – E °‘ (donor)
Aoxidised
A reduced
OXIDATION
REDUCTION
B reduced e-
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 (-ve DG)
if DE°‘ is +ve
ie: when E °' of the acceptor > E °' of the donor
Electrons spontaneously flow from low  high reduction potentials

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

27. thermodynamics of the ETChain
NAD accepts e- and H+ from fuel NADH
NADH donates e- and H+ to ETC
Hydride ion = 2e + H+
Accepts e- from fuel
Oxidised reduced
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
½ O2 + 2H+ + 2e-
H2O
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
½ O2 + 2H+ + 2e-
H2O
DE °‘= 1.13V DGo' = – 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
Intramembrane domains of cytochrome oxidase (complex IV) consist mainly of transmembrane a-helices.The

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. Has 4 metal ‘redox centers’
CuA (=2 Cu atoms)
haem a (Fe)
haem a3, (Fe)
CuB 
Haem a3 and CuB in subunit I: very close proximity (only 5 angstroms apart)  form a single ‘binuclear complex’
Ions in close proximity
= binuclear complex

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

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

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

38. The tricky bit!! 4e- are rearrangedO
Fully oxidised e- H+ O e- e- e- O
Fe2+ Cu+ e-
Tyr O H O- H
Fe3+ Cu2+
Fe2+ Cu+ O
Tyr
Tyr O H O H H O H
Fe2+ - 2e-  Fe4+ Cu e-  Cu2+
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
Tyr-OH - 1e- -H+  Tyr-O.
Fe2+ - 2e-  Fe4+ Cu e-  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
Fully oxidised e- H+ O e- e- e- O
Fe2+ Cu+ e-
Tyr O H O- H
Fe3+ Cu2+
Fe2+ Cu+ O
Tyr
Tyr O H O H H O H H O
Fe2+ - 2e-  Fe4+ Cu e-  Cu2+ Tyr-OH - 1e- -H+  Tyr-O. H H e- e- e- O- e-
O2- e-
O2-
Fe4+ Cu2+
Fe4+ Cu2+ e- e-
Tyr
Tyr O H O H e-
And more H+  H2O H

41. 4th e- passed via h3CuA Regenerates Fe3+: Completed cycle!
Fully oxidised e- H+ O e- e- e- O
Fe2+ Cu+ e-
Tyr O H O- H
Fe3+ Cu2+
Fe2+ Cu+ O
Tyr
Tyr O H O H H O H e- H H O
And one more H+
Fe2+ - 2e-  Fe4+ Cu e-  Cu2+ Tyr-OH - 1e- -H+  Tyr-O. H H e- e- e- O- e-
O2- e-
O2-
Fe4+ Cu2+
Fe4+ Cu2+ e- e-
Tyr
Tyr O H O e- H H

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

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. The individual reactions are:
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= kJ spontaneous
reduction
  ½ O2 + 2H+ + 2e- H2O DGo= kJ spontaneous
phosphorylation
  ADP   ATP DGo= kJ nonspontaneous
The net reaction is obtained by summing the coupled reactions,
ADP + NADH + ½ O2 + 2H+        ATP + NAD+ + 2 H2O
DGo= kJ spontaneous
Coupled non-spontaneous work