Lecture 19 Mitochondria, Membrane bound electron transfer and ATP synthesis

Question 1

Energy cannot be created or destoryed but it can be _____.

Answer 1

Energy can neither be created or destroyed, But it can be converted from one form to another

(oxidative phosphorylation: converting chemical energy into potencial enegry in a gradient, then reconverting it back into chemical energy)

Question 2

Who developed the theory behind oxidative phosphorylation?

“Coupling of Phosphorylation to Electron and Hydrogen Transfer by a Chemi-Osmotic type of Mechanism”Peter Mitchell (Nobel Prize 1978)

Answer 2

Peter Mitchell (Nobel Prize 1978)

Question 3

What is the summary of oxidative phosphorylation (2 steps)?

• The coupling of electron transfer and proton transfer generates _____.
• The proton-motive force is used to _____ : Protons move back into the matrix through _____ which _____ the formation of _____ from _____ and _____.

Answer 3

Step 1:

The coupling of electron transfer and proton transfer generates a gradient in proton concentration – an electrochemical gradient (proton-motive force)

Step2:

The proton-motive force is used to produce ATP: Protons move back into the matrix through ATP synthase which catalyses the formation of ATP from ADP and inorganic phosphate

Question 4

Explain this diagram:

Answer 4

• electrons are coupled with NADH, which are unstable due to the high energy state of NADH and want to reach a stable lower energy state
• the respiratory electron transport chain takes the electrons from NADH, and it uses the energy released as they try to get to water to cause structural changes in the proteins.
• These proteins bind protons at one side of the membrane and then they change shape while the electrons are moving through them which pushed these protons to the other side of the membrane which creates the gradient.
• when the gradient is established, these protons can go back, like a turbine, as the force of the protons trying to go down a concentration gradient rotates the atpase and then atp is synthesised.

Question 5

What is the relation between oxygen consumption by electron transport chain and ATP consumption?

Answer 5

As you hold your breath you see a decline in the consumption of ATP, and it goes up again when you breathe again.

The 2 are intrinsically connected

Question 6

Electrons move from (+ to -) or (- to ?)?

Answer 6

Electrons move from negative to positive.

Question 7

What are redox potencials?

Answer 7

its the potencial to give away an electron (E’₀)

Question 8

Electrons in an atom occupy a shell, which are normally in pairs. If unpaired, they are unhappy in a very high energy state which makes it likely to do what?

Answer 8

It makes the unpaired electrons likely to leave the shell to reach a low energy state. (oxidation)

Question 9

What is the difference between something being oxidised and reduced?

Answer 9

Question 10

What do we split redox reactions into?

Answer 10

redox couples

Question 11

What are the redox couples?

Answer 11

Question 12

What are Oxidation-reduction potentials (redox potentials)?

Answer 12

• Potential to donate electrons to the redox couple, 1/2 H₂ ↔ H⁺ + e^- (measured as V) in an electrical cell. (using the standard of hydrogen)
• Potential = V at half reduction (1M each)
• can workout the likelyhood of each chemical will give or take an electron.

Question 13

Redox potencials:

In this senario with the first solution being less stable and at a higher energy state, what will happen?

Answer 13

• electrons will leave A, and go to H, making more H_2, reaching a lower energy state
• the electrial current will be measured as negative (V)

Question 14

• If the electrons move towards H₂, a (+/-) electrical current will be measured.
• If the electrons move away H₂, a (+/-) electrical current will be measured.

Answer 14

• If the electrons move towards H₂, a - electrical current will be measured.
• If the electrons move away H₂, a + electrical current will be measured.

Question 15

Would the redox potencial of Na be positive or negative?

Answer 15

Negative

Question 16

Would the redox potencial of F be positive or negative?

Answer 16

Positive

Question 17

Knowing the redox potencials of Na and F, how can we calculate whether electrons will flow from one to another?

RP (Na) = -2.7V

RP (F) = +2.866V

Answer 17

E°(cell) = E°cathode - E° anode

= 2.866 - (-2.7)

= 5.586V (VERY energetically favorable)

Question 18

Explain redox couples in respiration

Answer 18

• measuring redox potencial of NADH against H₂, the value is negative, meaning it will give away electrons.
• measuring redox potencial of O₂ against H₂, the value is postive, meaning it will want to take electrons.
• alot of energy produced from respiration process when measuring the E ́o (cell) when tranfering electrons from NADH to O₂ to produce water

Question 19

What are the complexes composed in the respiratory electron-transport chain?

Answer 19

• Complex I (NADH dehydrogenase)
• Complex III (cytochrome bc1)
• Complex II (succinate dehydrogenase)
• Complex IV (cytochrome c oxidase)

Question 20

How does the respiratory electron-transport chain work?

Answer 20

1. complex I (NADH dehydrogenase)
   
   • takes electrons from NADH
   • electrons move through redox centers in complex I
   • while trying to get to the next complex (III), they force the protein to move –> causing the binding of protons outside of the membrane (matrix), suffling the electrons across the membrane to the intermembrane space
   • the electrons are now on Ubiquinone, (liquid soluble electron carrier molecule, - can be reduce or oxidized, it is hydrophobic) - which trasnfers electrons between complexes, the electrons are now transfered to complex III
2. complex III (cytochrome bc₁)
   
   1. more proton pumping
   2. electrons pass to soluble electron carrier protein in the innermembrane space of the mitochondria called cytochrome c
   3. cytochrome c carries electrons to complex IV
3. complex II (succinate dehydrogenase)
4. complex IV (cytochrome c oxidase)
   
   1. electrons try to move from cytochrome c to complex IV to O₂, down a large energy gradient which shifts groups (side chains) inside the complex which causes the pumping of protons
   2. leading to the reduction of O₂ to water

Question 21

What does Ubiquinone do?

Answer 21

connects the complexes, and caries H⁺ across the membrane

Question 22

Explain what is happening to Ubiquinone:

Answer 22

• one delocalised electron being transfered, creating a negative charge, that allows it to bind to a proton at the second step
• then there is the second electron donation bring in another negative charge
• when ubiquinone gains an electron it also gains a proton, which is apart of the mechanism for pumping

Question 23

Explain what is happening to Ubiquinone:

Answer 23

• electrons are reduced at complex I at the n (negative) side of the membrane, while protons are being taken away
• Ubiquinone moves through the bilayer to complex III, giving the electron away on the p side of the membrane
• as soon as the electrons are lost (oxidation), the Ubiquinone doesnt have a negative charge any more and the protons dissociate

Question 24

Proteins are very bad at binding electrons, so what do they usual contain?

Answer 24

redox centers (making use of transition metals)

• transition metals can be in many states, have d orbitals
  
  • electrons move between them​
    
    • can have Fe³⁺ or Fe²⁺ (having an extra electron)
      
      • apart of haem molecules
    • can have Fe - Su clusters (electrons are more delocalised over clusters)
    • also Cu centers

Question 25

What does Complex I (NADH dehydrogenase) do?

Answer 25

* catalyses transfer of electrons from NADH to ubiquinone * transports 4 H⁺ per pair of electrons

Question 26

Electron-transport chain: Sequence of electron carriers. Explain the diagram:

Answer 26

* y axis: high energy (**-** E'o) to low energy (**+** E'o) * Electron carriers are arranged from negative to positive redox potential * **plotting all of the centers which tells us the likely direction electrons are going to move**

Question 27

**Coupling sites: Complex I** Explain the diagram:

Answer 27

Complex I is a coupling site: electron transfer is accompanied by translocation of H+ across the inner mitochondrial membrane * NADH is transfered to Q (quinone) with the sufficient free energy availble to pump protons across IMM, which allows electrons to be transfered between them * the corresponding energy release used to cause structural changes that pump protons * reduced quinone

Question 28

What does Complex III (cytochrome bc1) do?

Answer 28

* catalyses transfer of electrons from ubiquinol to cytochrome c * transports 4 H+ per pair of electrons

Question 29

Coupling sites: Complex III Explain the diagram:

Answer 29

Complex III is a coupling site: H+ translocation is coupled to the oxidation of UQH₂ to UQ. Transition from 2 e- carrier (UQH₂) to a 1 e- carrier (Cyt c) * Q through haem to CC * complex II cannot pump protons because the electrons are going in as FADH which has the same redox potencial as the Q, so there is no energy to do pumping*

Question 30

What does Complex IV (cytochrome c oxidase) do?

Answer 30

* catalyses transfer of electrons from cytochrome c to O₂ * transports 2 H+ per pair of electrons

Question 31

Coupling sites: Complex IV Explain the diagram:

Answer 31

Complex IV is a coupling site: Electrons are transferred one at a time (multiple steps), resulting in the accumulation of reducing equivalents in the complex. Reduction of O₂ produces metabolic water. * very large drop, as electrons move through Cu centers and haems, it causes structural changes in the protein which ends up pumping protons

Question 32

The proton gradient (proton motive force) is used to synthesize \_\_\_\_\_.

Answer 32

proton gradient (proton motive force) is used to synthesize **ATP by the ATPase (complex V)**

Question 33

How can the proton gradient can be harvested to phosphorylate ATP?

Answer 33

* A proton gradient is a chemical gradient * A proton gradient is also an **electrical gradient** ## Footnote *The proton gradient is created using the electron transport chain to pump protons*

Question 34

Explain harvesting the gradient: the ATP synthase

Answer 34

* protons move from one side of the gradient at the top through a rotating kindof turbine (*right picture: blue part of molecule, left picture: stick in the mushroom rotates*) * this distorts the shape * the physical movement of the proteins drives the synthesis of ATP * pushing ATP and phosphate close together that it is a low energy state when ATP is formed

Question 35

What drives the rotation?

Answer 35

* Fo a subunit contains two separate half-channels * A proton from one half-channel binds to the carboxyl group of Asp61 on a c subunit * This causes a change in the c subunit and the c ring rotates * Another c subunit contacts the other half-channel, releasing its proton from Asp61 to the other side

Question 36

Rotational catalytic mechanism of the ATPase: explain the structure

Answer 36

There a 3 active sites (one on each β-subunit) They all have different conformations, depending on the orientation of the γ- stalk Either. • Open (O): cannot bind ATP or ADP * Loose (L): high affinity for binding ADP and P * Tight (T): pushes ADP and P together, forcing the synthesis of ATP

Question 37

Rotational catalytic mechanism of the ATPase: what happens when the *y* *subunit* moves around?

Answer 37

Movement of the γ-stalk through 120° therefore synthesizes 1 ATP by changing one β-subunit from L to T conformation. Complete rotation through 360° will therefore move each β- subunit through a complete O\>L\>T cycle, synthesizing 3 ATP

Question 38

How is ATPase regulated?

Answer 38

ATPase is regulated **by the availability of ADP (cell energy balance)** * Regulation of oxidative phosphorylation is entirely demand (ATP) driven * *if you dont have ADP, the ATPase doesnt work* * *if the ATPase doesnt work, the proton gradient is very high and too hard for the electron transport chain to push protons across*

Question 39

_Summary_: The coupling of electron transfer and proton transfer across the IMM

Answer 39

* The “reducing power” of NADH and FADH2 drives electron transfer reactions that finally reduce O₂, yielding water * These electron transfer processes cause structural changes in proton pumps and so are coupled to the pumping of protons (hydrogen ions) * This proton gradient (proton motive force) is used to synthesize ATP by the ATPase (complex V) * ATPase is regulated by the availability of ADP (cell energy balance)