1. MEMBRANE-BOUND ELECTRON TRANSFER AND ATP SYNTHESIS (taken from Chapter 18 of Stryer)

2. FREE ENERGY – MOST USEFUL THERMODYNAMIC CONCEPT IN BIOCHEMISTRY
Living things require an input of free energy for 3 major purposes
Mechanical – Muscle contraction and other cellular movement
Active transport of molecules and ions
Synthesis of macromolecules and other biomolecules from simple precursors

3. First law of thermodynamics
“Energy can be neither created nor destroyed”
But, it can be converted from one form into another

4. Free energy for these processes comes from the environment   Phototrophs - obtained by trapping light energy   Chemotrophs – energy by oxidation of foodstuffs   Free energy donor for most energy requiring processes is Adenosine triphosphate (ATP)

5. Large amounts of free energy is liberated when ATP is hydrolysed to ADP + Pi or AMP + PPi   ATP is continuously formed and consumed   Phototrophs harness the free energy in light to generate ATP - Photosynthesis   Chemotrophs form ATP by oxidation of fuel molecules – Oxidative phosphorylation

6. OXIDATIVE PHOSPHORYLATION
Glucose is converted to pyruvate
And under aerobic conditions undergoes oxidative decarboxylation to form AcCoA which is then oxidised to CO2 by the citric acid cycle

7. Stages of Catabolism

8. Activated Carriers
Glycolysis
Citric Acid Cycle

9. These pathways along with fatty acid oxidation produce energy rich molecules NADH and FADH2 as well as small amounts of ATP
 Chemotrophs derive energy from oxidation of fuel molecules and in aerobic organisms the ultimate electron acceptor is O2
Electron is not transferred directly
Electron is transferred through special carriers, Pyridine nucleotides
  Electron acceptor Electron donor
  NAD+ NADH
  FAD FADH2

10. Respiratory electron transfer is the transfer of electrons from the NADH and FADH2 (formed in glycolysis, fatty acid oxidation and the citric acid cycle) to molecular oxygen, releasing energy.
Oxidative phosphorylation is the synthesis of ATP from ADP and Pi using this energy.
Both processes are located on the IMM

11. Mitochondrion

12. Outer membrane Inner membrane Permeable (12000da)
Porin – 30-35kd pore forming protein
Inner membrane
Impermeable all ions and polar molecules
Possess family of transporter molecules (for ATP/ADP , Pi , pyruvate, citrate , etc.).
Matrix side (N-negative), cytosolic side (P-postive)
Porins are beta barrel proteins that cross a cellular membrane and act as a pore through which molecules can diffuse.[1] Unlike other membrane transport proteins, porins are large enough to allow passive diffusion, i.e., they act as channels that are specific to different types of molecules. They are present in the outer membrane of Gram-negative bacteria and some Gram-positive bacteria of the group Mycolata (mycolic acid-containing actinomycetes), the mitochondria, and the chloroplast.

13. Mitochondria are the result of an Endosymbiotic event
Organelles contain their own DNA which encode 13 respiratory chain proteins
Many proteins encoded by cell nuclear DNA
Cells depend on organelle for oxidative phosphorylation , mitochondrion depend on cell for their very existence
Suggested that all existing mitochondria are derived from bacterial Rickettsia prowazekii

14. Oxidative phosphorylation is conceptually simple and mechanistically complex.
Flow of electrons from NADH and FADH2 to O­2 occurs via protein complexes located in the IMM
Leads to the pumping of protons from the matrix to the cytosol across the IMM.
ATP is synthesised when protons flow back into the matrix via a protein complex in the IMM.

15. An example of energy coupling via an electrochemical gradient across a membrane.

16. REDOX POTENTIAL AND FREE ENERGY CHANGES The energy stored in ATP is expressed as the phosphoryl transfer potential which is given by G­o for hydrolysis of ATP (-7.3kcal/mol) The electron transfer potential of NADH is represented as Eo the redox potential ( or reduction potential or oxidation-reduction potential) which is an electrochemical concept. Redox potential is measured relative to the H+: H2 couple which has a defined redox potential of 0V (Volts).
Electron transfer potential is converted into phosphoryl transfer potential

17. Redox couples
Substance can exist in oxidised form X and reduced form X- Reduction potential can be determined from the above apparatus. Basically comparing the reduction potential of this couple against a standard which contains. Electrons travel across the agar bridge whilst the

18. A negative redox potential means that a substance has a lower affinity for electrons than H2 . A positive redox potential means a substance has a higher affinity for electrons than H2. NAD+/ NADH at -0.32V is a strong reducing agent and poised to donate electrons 1/2 O2/ H2O at +0.82V is a strong oxidising reagent and poised to accept electrons /2 O NADH +H H2O NAD The difference (Eo = 1.14V) is equivalent to kcal/mole.

20. Electrons can be transferrred between groups that are not in contact
How can electrons be carried between proteins. These groups are usually buried within the structure of a protein. The protein environmemnt increaeses the efficiency of the transfer of an electron. Groups are typicfally separtaed by 15A beyond their Van der Waals contact distance

21. THE RESPIRATORY ELECTRON TRANSFER CHAIN CONSISTS OF THREE PROTON PUMPS LINKED BY TWO MOBILE ELECTRON CARRIERS I
Electrons are transferred from NADH to O2 by a chain of three large transmembrane respiratory chain protein complexes II
III
These are the proteinsd involved in electron transfer. There are three transmembrane proteins that act as prton pumps andf there are two mobile carriers that are responsible for transfer between these three complexes. IV

22. These are
a) Complex I also known as
NADH-Ubiquinone (UQ) oxidoreductase
NADH-Q reductase
b) Complex III also known as
Ubiquinol (UQH2)-Cytochrome c oxidoreductase
Cytochrome reductase 
c) Complex IV also known as
Cytochrome c- Oxygen oxidoreductase
Cytochrome oxidase

23. Q- coenzyme Q or ubiquinone
If we look at each in more detail, we can see that they contain within them prosthetic groups which are responsible for the transport of electrons through the protein. They are either flavins, iron sulfur clusters or heme or copper centres
Q- coenzyme Q or ubiquinone

24. Complex I NADH-Q Oxidoreductase

25. NADH-Q reductase
NADH transfer of e- to flavin mononucleotide to produce FMNH2
e- from FMNH2 transferred to iron sulfur clusters
e- from iron sulfur (Fe-S) clusters shuttle to coenzyme Q (ubiquinone)
Results in pumping of 4 H+ out of matrix
NADH + Q + 5H+matrix NAD+ +QH H+cytosol

26. Complex II Succinate Q reductase
FADH2 already part of complex, transfers electrons to Fe-S centres and then to Q
This transfer does not result in transport of protons

27. Complex III Q-cytochrome c Oxidoreductase
Heme bl is low affinity and heme b h is high affinity

28. Q-cytochrome c Oxidoreductase
Transfers e- from QH2 (2 e- ) cytochrome c (1 e- ) via heme
Mechanism known as Q cycle
QH2 + 2Cyt cox + 2H+matrix Q +2Cyt cred + 4H+cytosol

29. Q Cycle QH2 QH2 + 2Cyt cox + 2H+matrix Q +2Cyt cred + 4H+cytosol
Two electron carrier to a one electron carrier
QH2 + 2Cyt cox + 2H+matrix Q +2Cyt cred + 4H+cytosol

30. Complex IV Cytochrome c Oxidase

31. Cytochrome c Oxidase

32. Proton transport by cytochrome c oxidase

34. Electrons are carried from Complex I to Complex III by UQH2, the hydrophobic quinol (reduced quinone) diffuses rapidly within the IMM.
Electrons are carried from Complex III to Complex IV by cytochrome c, a small hydrophilic peripheral membrane protein located on the cytosolic or P side of the IMM.
Complex II (Succinate-UQ oxidoreductase) is membrane bound and contains the FADH2 as a prosthetic group . So electrons from FADH2 feed in to UQH2.
These respiratory chain complexes contain redox groups to carry the electrons being transferred through them. These are flavins, iron-sulfur clusters, haems and copper ions.

36. PROTON PUMPS AND THE ATP SYNTHASE
The free energy change of the reactions catalysed by Complexes I, III and IV is large enough for them to pump protons from the matrix or N side of the IMM to the cytosolic or P side of the IMM.
There is not enough energy released in Complex II, so no proton pumping occurs in this complex.

37. OXIDATION AND PHOSPHORYLATION ARE COUPLED BY A PROTON-MOTIVE FORCE
This is the chemiosmotic hypothesis put forward by Peter Mitchell in 1961.
Transfer of electrons from NADH (or FADH2) to oxygen leads to the pumping of protons to the cytosolic side of the IMM.
The H+ concentration (pH) becomes higher (lower pH) on the cytosolic side, and an electrical potential (membrane potential) with the cytosolic side of the IMM positive is generated

39. So a proton-motive force (p) is generated which consists of both a pH and a .
Mitchell proposed that this proton-motive force drives the synthesis of ATP by another transmembrane protein complex, as the protons return back across the IMM through this protein complex.
This protein complex is called the ATPase (because like any enzyme it is reversible and was first discovered by it’s ability to hydrolyse ATP)
It’s preferred name is the ATP synthase.

40. Structure of ATPase
10-14 c subunits

41. ATP synthase nucleotide binding sites are not equivalent
Alpha and beta chains both bind nucleotides but it is the beta subunit that participates in ATP synthesis. Each beta subunit exists in a different conformation termed O – open , L – loose and T – tight. In open form ATP released and ADP and Pi then bind and cycle repeats. These changes are driven by the rotation of the central protein lambda which enables each subunit to exist in each of the states as it turns through 120 degrees

43. It is now thought that the proton-motive force induces a conformational change in the ATP synthase, which allows the release of tightly bound ATP (the product) from the enzyme, and thus catalyses ATP synthesis.
Ultimately each 360 degree turn will produce An ATP in each of the three beta subunits
So this is an example of energy coupling via an activated protein conformation.

44. C-ring
H+ in
How is this rotation achieved? Each c subunit contains two half channels. An aspartate lies at the centre of the c-ring subunit.
H+ out

45. Protonated aspartate prefers to reside in hydrophobic environmemnt of the membrane. Thus the c ring rotates clockwise towards the lipid membrane environmwemnt. It continues to turn until it reaches this position where now the proton is in the hydrophilic environment of the matrix environmemnt and the proton moves into the matrix

46. Rotates 360º
Producing 3 molecules of
ATP
Thus 10 protons required
Requires 3 protons per
molecule of ATP

48. THE COMPLETE OXIDATION OF GLUCOSE YIELDS ABOUT 30 ATP

49. Net Yield per glucose
Glycolysis ATP
Citric Acid cycle ATP (GTP)
Oxidative phosphorylation ~26 ATP
Most of the ATP is generated by oxidative phosphorylation

50. POWER TRANSMISSION BY PROTON GRADIENTS: A CENTRAL MOTIF OF BIOENERGETICS
Proton gradients power a variety of energy-requiring processes i.e.

51. IT IS EVIDENT THAT PROTON GRADIENTS ARE A CENTRAL INTERCONVERTIBLE CURRENCY OF FREE ENERGY IN BIOLOGICAL SYSTEMS.
THE RATE OF OXIDATIVE PHOSPHORYLATION IS DETERMINED BY THE NEED FOR ATP
Under most physiologic conditions, electron transfer is tightly coupled to phosphorylation. Electrons do not usually flow through the electron transfer chain unless ADP is simultaneously phosphorylated to ATP.
Oxidative phosphorylation and thus electron transfer require a supply of
NADH O2
ADP and Pi

52. The most important factor controlling the rate of oxidative phosphorylation is the level of ADP Regulated by the energy charge. This regulation of the rate of oxidative phosphorylation by the ADP level is called respiratory control.