CC2 Flashcards
What does the chemiosmotic theory state about proton pumping in energy-transducing membranes?
- 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.
What are the P-phases and N-phases in gram-negative bacteria, mitochondria, and chloroplasts?
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)
How can mitochondria and gram-negative bacteria be prepared to study their energy-transducing membranes?
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.
What is a submitochondrial particle?
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.
What happens when an ion motive force cannot be generated by an ETC? Give an example.
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.
What equation did Mitchell use to define the PMF?
Δp= - (electrochemical potential of protons)/F
What is the equation for an ion moving from N to P that is affected by both concentration and electrical gradients?
ΔG=-zFΔψ+RTln([X^(z+) ]_P/[X^(z+) ]_N )= 〖Δμ〗_X^(z+)
How does permeability of the membrane to protons affect the driving force?
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.
How do buffers impact the PMF?
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.
What are the major buffers in cells and which one is the strongest buffer?
- Proteins
- Phosphate
- Bicarbonate
The cell is full of proteins, so these are the strongest buffer.
How can membrane potentials be generated experimentally?
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.
What is the mitochondrial ATP cycle?
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.
How does the mitochondrial transport cycle impact the membrane potential? (Why is the energetic cost of the ATP cycle one proton?)
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.
Why is the ATP:ADP+Pi ratio massively displaced from equilibrium in living cells?
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.
Why is the driving force of ATP synthase stronger in the cytoplasm than in the matrix?
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.
What are the 4 essential tests Mitchell proposed for the chemiosmotic hypothesis?
- ETCs should pump protons.
- ATP synthase should be reversible i.e., work as an ATPase that translocates protons.
- Energy-transducing mechanisms should have a low effective proton conductance.
- Energy-transducing membranes should possess specific exchange carriers to permit metabolites to permeate in the presence of a high negative inside membrane potential.
How does the chemiosmotic mechanism explain the action of uncouplers?
Uncouplers break the link between ATP synthesis and electron transfer. E.g., uncoupling proteins allow brown adipose tissue mitochondria to generate heat.
Describe the oxygen pulse experiment.
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.
Describe the acid-bath experiment.
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.
Describe the acid-bath with rapid mixing experiment.
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.
Describe the ATP-pulse experiment.
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.
Describe how chemiosmotic circuits showed that ATP generation doesn’t require physical contact between ATP synthase and PMF-generating proteins.
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.
How does the chemiosmotic mechanism account for respiratory control?
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.
What are flavin cofactors?
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.
