CC2 - How do cells generate PMF?

Question 1

What does the chemiosmotic theory state about proton pumping in energy-transducing membranes?

Answer 1

• Energy-transducing membranes contain pairs of proton pumps and in each case, the primary pump utilizing either electrons or photons pumps protons from the N (negative) phase to the P (positive) phase.
• In contrast to the variety of primary proton pumps, all energy-transducing membranes contain a highly conserved secondary proton pump termed the ATP synthase.
• The essence of the chemiosmotic theory is that the primary proton pump generates a sufficiently large electrochemical gradient of protons to force protons back through the secondary pump so that it reverses and synthesizes ATP.

Question 2

What are the P-phases and N-phases in gram-negative bacteria, mitochondria, and chloroplasts?

Answer 2

Gram-negative bacteria:
P-phase - periplasm
N-phase - cytoplasm
(protons pumped outwards from cytoplasm to periplasm)

Mitochondria:
P-phase - intermembrane space
N-phase - matrix
(protons pumped outwards from matrix to IM space)

Chloroplast:
P-phase - thylakoid lumen
N-phase - stroma
(protons pumped inwards from stroma to lumen)

Question 3

How can mitochondria and gram-negative bacteria be prepared to study their energy-transducing membranes?

Answer 3

Mitochondria:
- Sonication to break up the inner membrane, causing self-sealing in an inverted orientation i.e., positive-inside. This is called the submitochondrial particle.

Gram-negative bacteria:
- Lysozyme to break down cell wall and osmotic shock to rupture the outer membrane i.e., positive-outside.
- French press ruptures the cell so it self-seals with a positive-inside.

Question 4

What is a submitochondrial particle?

Answer 4

A submitochondrial particle (SMP) is a term used to describe a preparation of mitochondrial membranes that have been disrupted or solubilized using detergents or other agents.
SMPs are useful tools for studying mitochondrial function, as they allow researchers to isolate and manipulate specific components of the mitochondrial membrane in a controlled manner.

Question 5

What happens when an ion motive force cannot be generated by an ETC? Give an example.

Answer 5

Ion-motive gradients are so fundamental to cellular function that where they cannot be produced by electron transport chains, they are generated at the expense of ATP hydrolysis.
E.g., fermenting bacteria use ATP synthase is reverse to pump protons out of the cell to generate ion gradients for membrane transport processes.

Question 6

What equation did Mitchell use to define the PMF?

Answer 6

Δp= - (electrochemical potential of protons)/F

Question 7

What is the equation for an ion moving from N to P that is affected by both concentration and electrical gradients?

Answer 7

ΔG=-zFΔψ+RTln([X^(z+) ]_P/[X^(z+) ]_N )= 〖Δμ〗_X^(z+)

Question 8

How does permeability of the membrane to protons affect the driving force?

Answer 8

As you approach 200mV, the permeability of the membrane to protons gets much larger. If the proton motive force was larger than 200mV, the protons pumped out would immediately move back across the membrane and so you couldn’t store their energy. Thus, 200mV is the maximum compromise.

Question 9

How do buffers impact the PMF?

Answer 9

Cell compartments are heavily buffered, meaning their pH is clamped so that when protons are being moved around the cell, the pH doesn’t differ significantly but the charge component does. Hence, the electrical component contributes more significantly to the electrochemical potential, and thus the PMF.

Question 10

What are the major buffers in cells and which one is the strongest buffer?

Answer 10

• Proteins
• Phosphate
• Bicarbonate

The cell is full of proteins, so these are the strongest buffer.

Question 11

How can membrane potentials be generated experimentally?

Answer 11

Based on the equation to determine the electrochemical potential, if the electrochemical potential is 0, the electrical term is the exact equal but opposite of the concentration term.
By using valinomycin to artificially set up a membrane potential of potassium ions, the ions will keep moving down their concentration gradient until the charge gradient is equal and opposite in magnitude to the concentration gradient i.e., the electrochemical potential is 0.

You can therefore work out the membrane potential based on the ratio of ions on the inside and outside of the cell. This can then be used to set the size of the charge gradient wanted to be imposed on the membranes.

Question 12

What is the mitochondrial ATP cycle?

Answer 12

The mitochondrial ATP cycle refers to the process by which ATP is produced within the mitochondria of eukaryotic cells and transported back into the cytoplasm. The resulting ADP and Pi from ATP hydrolysis are then transported back into the mitochondria for ATP synthesis.

Question 13

How does the mitochondrial transport cycle impact the membrane potential? (Why is the energetic cost of the ATP cycle one proton?)

Answer 13

The movement of ADP and Pi goes against the charge gradient across the membrane and is opposed by the proton motive force. To solve this, ADP3- movement is coupled with the export of ATP4- so that the net charge movement is favorable. This then decreases the charge component of the proton motive force because we’ve decreased the amount of positive charge on the outside of the membrane.

Pi- is co-transported with one proton (via a phosphate carrier) such that the charges cancel and the net charge movement is 0.

ADP and Pi must be kept at a 1:1 ratio, and so the total energetic cost to the proton motive force of completing the ATP cycle once is the combined cost of the adenine nucleotide transporter AND the phosphate carrier. This energetic cost is shown to be the exact same as moving a single proton down its electrochemical gradient.

Question 14

Why is the ATP:ADP+Pi ratio massively displaced from equilibrium in living cells?

Answer 14

In order for ATP to function as a high-energy currency in the cell, it must exist at a concentration that is significantly higher than its precursors, ADP and Pi. This creates a large free energy difference between ATP and its hydrolysis products, which allows ATP to serve as a readily available source of energy for cellular processes.

Question 15

Why is the driving force of ATP synthase stronger in the cytoplasm than in the matrix?

Answer 15

For the cytoplasm, you take into account the transport of ATP into the cytoplasm (increasing its concentration) and the transport of ADP into the matrix (reducing its concentration), despite the fact that ATP is being broken down into ADP in the cytoplasm. This pushes the mass action ratio further to the left (reflected in the ATP:ADP ratio). Instead of the mass action ratio being eight orders of magnitude away from equilibrium, as it is in the matrix, it’s now 10 orders away.

Question 16

What are the 4 essential tests Mitchell proposed for the chemiosmotic hypothesis?

Answer 16

1. ETCs should pump protons.
2. ATP synthase should be reversible i.e., work as an ATPase that translocates protons.
3. Energy-transducing mechanisms should have a low effective proton conductance.
4. Energy-transducing membranes should possess specific exchange carriers to permit metabolites to permeate in the presence of a high negative inside membrane potential.

Question 17

How does the chemiosmotic mechanism explain the action of uncouplers?

Answer 17

Uncouplers break the link between ATP synthesis and electron transfer. E.g., uncoupling proteins allow brown adipose tissue mitochondria to generate heat.

Question 18

Describe the oxygen pulse experiment.

Answer 18

Mitochondria were bathed in anoxic medium, allowing for defined pulses of oxygen to be inserted. A pH electrode was used to measure the pH component of the PMF in the extra-mitochondrial space. Hence, to equate this to be the entire PMF, the electrical component had to be dissipated through the use of valinomycin.
The amount of oxygen injected were only large enough to elicit a change in pH, but not membrane potential.
After each pulse of oxygen, protons would move into the matrix through ATP synthase. Once the oxygen has run out, protons would start to leak back through ATP synthase (now acting as an ATPase), increasing the pH.
This showed that ETCs generate a PMF.

Question 19

Describe the acid-bath experiment.

Answer 19

Thylakoids were bathed in acidic medium until the interior and exterior pHs were at equilibrium. These thylakoids were then moved to a high pH medium that contained ADP and Pi. These conditions set up a proton gradient across the thylakoid membrane. Protons would thus move out of the thylakoids, driving the synthesis of ATP. Interestingly, this could be achieved in the dark despite photosynthesis requiring photons. Thus, the PMF drives ATP synthesis, not electron transfer.

Question 20

Describe the acid-bath with rapid mixing experiment.

Answer 20

Repeat the acid-bath experiment and measure the rate of ATP synthesis via rapid mixing techniques. These experiments showed that the rate of ATP synthesis driven by the PMF is essentially the same as the rate of ATP synthase that’s driven by electron transfer.
Thus, the PMF is kinetically competent to drive ATP synthesis.

Question 21

Describe the ATP-pulse experiment.

Answer 21

For the PMF to be thermodynamically competent to drive ATP synthesis, the energy stored in the PMF had to be equal to the ΔG for the phosphorylation potential of ATP in the matrix.

To determine this, they calculated the ratio of H+:ATP in the ATP synthase reaction by repeating the oxygen-pulse experiment but using ATP-pulses instead. ATP synthase will hydrolyze the ATP and pump protons out of the mitochondria to generate a PMF. This allows for the number of protons extruded per molecule of ATP to be determined. This showed s = 2.7.

To determine the PMF, permeable anions or acids were allowed to equilibrate across a membrane. The resulting concentrations were then used to calculate the electrical and pH gradients, respectively. This gave a PMF of -19.3kJ mol-1.

Question 22

Describe how chemiosmotic circuits showed that ATP generation doesn’t require physical contact between ATP synthase and PMF-generating proteins.

Answer 22

Investigators created artificial vesicles to mimic the membranes involved in chemiosmosis. To these vesicles, they added a light-driven proton pump, bacteriorhodopsin, from bacteria.
When illuminated, the pH of the external solution increased, meaning protons were being pumped into the vesicles and creating a proton gradient.
If ATP synthase from mitochondria of bovine cells is also incorporated into vesicles, the vesicles form ATP. The investigators concluded that ATP synthase, acting as a proton channel, is necessary for ATP synthesis.
There can’t be any interactions between the two proteins because they are separated by billions of years of evolution.

Question 23

How does the chemiosmotic mechanism account for respiratory control?

Answer 23

The chemiosmotic mechanism explains how the proton motive force is used to produce ATP, and also how respiratory control is achieved. When energy demands increase, cells can increase their rate of respiration by increasing the activity of the ETC and pumping more protons into the intermembrane space. This increases the proton motive force and ATP production.

Conversely, when energy demands decrease, cells can reduce their rate of respiration by reducing the activity of the ETC and pumping fewer protons into the intermembrane space. This decreases the proton motive force and ATP production.

Therefore, the chemiosmotic mechanism provides a way for cells to regulate their rate of respiration and ATP production in response to changing energy demands, thus accounting for respiratory control.

Question 24

What are flavin cofactors?

Answer 24

Flavin cofactors are a type of organic molecule that act as electron carriers in many biological reactions. They are derived from riboflavin (vitamin B2) and include two types: flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD).

FMN and FAD are both coenzymes that function as electron carriers in a variety of metabolic pathways, including cellular respiration and photosynthesis. They are capable of accepting and donating one or two electrons at a time, depending on the redox state of the molecule.

Question 25

Why is there a 14 angstrom practical limit to redox cofactor separation in proteins?

Answer 25

The probability of electron transfer occurring is dependent on the degree of overlap between the two wave functions. In biology, all electron transfer reactions occur within 14 angstroms, likely because biology doesn’t want the ETCs to be rate-limiting.
To ensure this electron transfer rate is maintained, biology reduces cofactor separation to less than 14 angstroms. This can be achieved by moving the donor and acceptor closer, or by adding another cofactor as an intermediate so that the two electron steps are less than 14 angstroms.

Question 26

How does the complex II protein environment tune the reduction potential of its bound cofactors?

Answer 26

Complex II contains three heme groups and a flavin adenine dinucleotide (FAD) cofactor that are involved in electron transfer reactions. The protein environment surrounding these cofactors can influence their electron transfer properties by stabilizing the reduced form of flavin, and hence making it a better electron acceptor. Otherwise, its reduction potential is far too negative for the reaction to ever work.

Question 27

What are secondary transporters, and why can’t protons be traditionally moved through these?

Answer 27

TM proteins that use electron transfer reactions to generate energy for conformational changes that results in the transfer of a molecule against its gradient.

Protons only exist as part of the solvent rather than as free entities so cannot travel via these methods.

Question 28

Describe the Grotthuss mechanism for proton transfer.

Answer 28

The basic idea behind the Grotthuss mechanism is that a proton can transfer from one molecule to another along a chain of hydrogen bonds. The process begins with the transfer of a proton from a donor molecule to an acceptor molecule, which occurs through the breaking and reforming of hydrogen bonds. Once the proton has been transferred, the acceptor molecule becomes the donor molecule, and the process can repeat itself along the chain.

One important feature of the Grotthuss mechanism is that it allows protons to move rapidly through the hydrogen-bonded network without requiring a significant amount of energy. This is because the breaking and reforming of hydrogen bonds that occurs during the process is energetically favorable and does not require the input of external energy.

Question 29

How are protons prevented from moving backwards in Grotthuss chains?

Answer 29

Conformational changes to disrupt Grotthuss chains.

Question 30

What is a redox loop?

Answer 30

A redox loop is a process in which a molecule undergoes a series of redox reactions, cycling between two different redox states. In the electron transport chain, electrons are passed from one electron carrier to another, with each carrier being oxidized and reduced in turn. This creates a redox loop that drives the synthesis of ATP, the main energy currency of the cell.

Question 31

Compare secondary transporters to redox loops in terms of PMF generations.

Answer 31

Conformational pump:
- Moves protons
- Water-filled channels and/or proton wires
- Requires conformational changes
- No fixed stoichiometry
- Can be adapted to generate a sodium motive force

Redox loops:
- Moves electrons
- Transmembrane electron transfer through redox centres
- No conformational changes
- 1 proton: 1 electron
- Cannot be adapted to generate a sodium motive force

Question 32

Why can’t complex II or ubiquinone oxidoreductase generate a protonmotive force?

Answer 32

In the case of Complex II, electrons are transferred from succinate to ubiquinone, generating ubiquinol. This process does not involve the transport of protons across the membrane and therefore does not contribute to the PMF.

Similarly, in the case of Complex III, electrons are transferred from ubiquinol to cytochrome c, but again, this process does not involve the transport of protons across the membrane.

In both cases, the redox potential isn’t sufficient to transfer a proton.

Question 33

What is a Rieske protein?

Answer 33

A Rieske protein (also known as a 2Fe-2S protein or Rieske iron-sulfur protein) is a type of protein that contains an iron-sulfur cluster with a unique structure. Rieske proteins play an important role in electron transfer reactions, as they are able to accept and donate electrons. In particular, they are involved in the transfer of electrons from ubiquinol to cytochrome c in Complex III.

Question 34

Describe the Q-cycle mechanism of complex III.

Answer 34

1) UQH2 oxidation at Qp - the first electron is transferred from UQH2 to the Rieske protein, releasing two protons into the cytoplasm and leaving the free radical semiquinone anion species at the Qp site. The second electron is transferred to the bL haem, which is also close to the P-face.
2) UQ reduction to the radical at Qn - the electron on bL passes to bH. UQ then binds in Qn, in the close vicinity of bH, allowing the transfer of the electron from the reduced bH to form a radical.
3) UQ radical is reduced to UQH2 - a semiquinone radical is now bound at the Qn site. Another UQH2 is oxidized at Qp, repeating step 1.
4) Formation of UQH2 from a radical - the second electron from this reaction now completes the reduction from the semiquinone radical to UQH2, the two protons required for this being taken up from the matrix. The UQH2 returns to the bulk pool and the cycle is completed.

Question 35

What is electron bifurcation?

Answer 35

In electron bifurcation, the energy from one electron is split and distributed to two or more molecules, with the high-energy electron being transferred to the most energetically demanding molecule. This process allows cells to conserve energy by using one molecule to drive multiple reactions that require energy input.

Question 36

How do Mitchelian loops explain why electron bifurcation occurs?

Answer 36

Mitchelian loops means that the acceptor from one side has to move to the other side to become the next donor, but cytochrome c cannot move across the membrane. Hence, the electrons are split using UQ/UQH2.

Question 37

Why does bL have a more negative reduction potential than bH?

Answer 37

The differences of reduction potentials between bL and bH is to allow for the favorable movement of electrons between the two haem groups, going against the unfavourability of the electron moving closer to the N phase which is more negative than the P phase.

Question 38

Why is the semiquinone radical only stabilized at the Qn site in complex III?

Answer 38

Semiquinone only has to be stabilized at Qn to allow for the two half reactions to be approximately equal in reduction potentials. This would be a waste of energy at Qp.

Question 39

How is the second electron from bifurcation at complex III prevented from reducing cytochrome c?

Answer 39

Rieske is over 14 angstroms away from cytochrome c when it accepts the first electron, preventing the second one from also binding. Affinities then control the movement towards cytochrome c to donate the electron, and now the second electron is too far away to move onto the Rieske so has to go to bL haem.

Question 40

Why is the actual coupling stoichiometry less than the potential stoichiometry at complex IV?

Answer 40

It ensures the reaction is essentially irreversible.
When oxygen is reduced, it goes through many reactive intermediates and so by making the reaction irreversible, the reaction is pushed further to the right thus making the time spent as these reactive intermediates reduced (protects the cells from oxidative species).

Question 41

Why isn’t the oxygen-reducing site in complex IV fully coordinated?

Answer 41

The oxygen-reducing site contains a haem binuclear center with vacant coordination sites. These vacant coordination sites allow for binding of intermediate species in the reaction.

Question 42

How are electrons transferred through the haem groups of complex IV?

Answer 42

1) CuA accepts electrons from haem c (cytochrome c), four times.
2) Transfers electrons to haem a, four times
3) Transfers electrons to haem a3, four times
4) Electrons reach the oxygen-reduction site to reduce oxygen species, combining with protons from the N-phase to allow for the end production of water.
This can only move one charge per electron, thus a proton pump must also be used.

Question 43

What are the 3 potential channels that protons can move through in complex IV?

Answer 43

K, D, and H channels.

Question 44

How is oxygen reduced at complex IV?

Answer 44

1. Oxygen binds the binuclear centre, a3-CuB.
2. The heme a3-CuB accepts 4 electrons from 4 molecules reduced cytochrome c, which are transferred to oxygen. This occurs in steps, producing radicals.
3. As the reduction of oxygen progresses, protons are simultaneously taken up from the matrix and pumped out. This is driven by electron transfer from cyt c to oxygen, creating an electrochemical gradient across the membrane.
4. The four electrons and 4 protons are coupled to form two molecules of water.

Question 45

How may proton pumping in complex IV be gated?

Answer 45

The charge on the binuclear center impacting the affinity for protons.

When an electron enters the center, the negative charge makes proton binding more favorable. Other protons entering from the active site causes a decrease in affinity, releasing the proton. Hence the proton can be moved from the N-side to the P-side.