Chemiosmosis Flashcards
What are some experimental evidence for the Chemiosmotic Hypothesis ?
- Coupling of electron transport and ATP synthesis
- Development of the ‘protonmotive force’ (pmf)
- Role of proton gradient in ATP synthesis
What 4 basic tenets is the Chemiosmotic hypothesis based upon ?
- An ion-impermeable inner membrane
- H+ translocating respiratory chain
- Reversible H+ translocating ATP synthase
- H+ or OH- -linked exchange diffusion system
What are the properties of the mitochondrion ?
- Outer membrane
- Freely permeable to small molecules (Mr < 5,000) and ions - Inner membrane
- Impermeable to most small molecules and ions including H+ - Matrix
- Contains enzymes for:
TCA cycle
β-oxidation
Amino acid oxidation
Many other enzymes
DNA and ribosomes
ATP, ADP, Pi, Mg2+, Ca2+, K+
Many soluble metabolic intermediates
What does the addition of weak HCl to mitochondria result in ?
An initial rapid acidification of the medium followed by an alkalination
Name the complexes of the ETC ?
Complex I - NADH dehydrogenase Complex II - Succinate dehydrogenase Complex III - Cytochrome bc1 complex Complex IV - Cytochrome aa3 oxidase Complex V - ATP synthase
Explain Complex I ?
- L-shaped, with one arm embedded in the inner membrane and the other extending into the matrix
- obligately coupled processes:
1. transfer of electrons from NADH to ubiquinone in mammals
2. transfer of four protons from the matrix to the intermembrane space
Where does Complex II function ?
In the citric acid cycle
Explain Complex II ?
- Couples the oxidation of succinate to fumarate at one site with the reduction of ubiquinone on the other.
- Electrons move (blue arrows) from succinate to FAD, then through the three Fe-S enters to ubiquinone.
- Electron transfer through Complex II is not accompanied by proton pumping across the inner membrane, although the QH2 produced by succinate oxidation will be used by Complex III to drive proton transfer
Explain Complex III ?
- Couples the transfer of electrons from dihydroquinone to cytochrome c
- Translocate protons from the matrix to the intermembrane space
- Complex III has two distinct binding sites for ubiquinone, QN and Qp, which correspond to the sites of inhibition by two drugs that block oxidative phosphorylation
1. Antimycin A, which blocks electron flow from cytochrome b to cytochrome c1
2. Myxothiazol, which prevents electron flow from QH2 to the Rieske iron-sulfur protein
Explain Complex IV ?
- carries electrons from cytochrome c to molecular oxygen, reducing it to H2O
- transport 2 molecules of protons (H+) at a time
Explain how the changes in reduction potential and free energy during the stepwise flow of electrons through the ETC ?
- Translocation of protons across the inner mitochondrial membrane
- Electrons pass through the multi protein complexes from those with a lower reduction potential to those with a higher (more positive) reduction potential (left scale), with a corresponding reduction in free energy (right scale).
- The energy released as electrons flow through three of the complexes is sufficient to power the pumping of H+ ions across the membrane, establishing a proton-motive force.
How are respirasomes formed ?
Although the four electron-transferring complexes can be separated in the laboratory, in the intact mitochondrion, the respiratory complexes tightly associate with each other in the inner membrane
How much protons does the respiratory chain translocate ?
10H+/2e-
Reduction of one molecular oxygen (O2) allow ?
Four electron flow in the ETC that translocates 20 protons (H+)
Explain Proton-motive Force ?
Electron transport chain proteins create the electrochemical proton gradient by one of these three mechanisms:
- actively transport protons across the membrane via relatively poorly understood mechanisms
- pass electrons to ubiquinone which picks up protons from the matrix to form dihydroquinone (QH2)
- protons released into the inter-membrane side when QH2 is oxidised
What are the two compartments of the proton motive force ?
- the chemical potential energy i.e. difference in pH (ΔpH) due to the difference in concentration of a chemical species (H+) in the two regions separated by the membrane
- the electrical potential energy (Δ) that results from the separation of charge when a proton moves across the membrane without a counterion
What is the 3rd tenet of the chemiosmotic hypothesis ?
A reversible proton translocating ATP synthase
Briefly explain the Chemiosmotic theory ?
- Energy needed to phosphorylate ADP is provided by the flow of protons down the electrochemical gradient
- The electrochemical gradient is established by transporting protons against the electrochemical gradient during the electron transport
What is evidence for the role of a proton gradient in the synthesis of ATP ?
The presence of mitochondria turned a weak acid solution to less acidic (i.e. alkalination).
Here, mitochondria are
i) equilibrated in a basic solution at pH 9.
ii) transferred to a neutral solution of pH 7.
iii) an increase of ATP concentration inside the Mt observed
A prediction of the chemiosmotic theory is ?
If the role of electron transfer in mitochondrial ATP synthesis is simply to pump protons to create the electrochemical potential of the proton-motive force, an artificially created proton gradient should be able to replace electron transfer in driving ATP synthesis.
What is proof that the F0 complex rotates ?
- Experiments by Masasuke Yoshida and Kazuhiko Kinosita
- Immobilized the F1 complex and attached an actin filament with a fluorescent dye to a c subunit of F0
- Examined under a microscope
How does ATP synthase work ?
Rotational catalysis is key to the binding-change mechanism for ATP synthesis and was proposed by Paul Boyer in 1980s after detailed kinetic and binding studies of FoF1 complex.
- Proton enters from the intermembrane space in the Foa subunit then binds to an amino acid in the Foc subunit that makes the Foc ring turn anti-clock wise.
- Proton binding to each c subunit turns the Foc carousel that in turn rotates the central shaft
- The three binding sites of F1 take turns in catalysing ATP synthesis
- Proton goes out into the matrix from the other proton half-channel in the Foa subunit
Explain Complex V: F-ATP synthase ?
- Located on the inner mitochondrial membrane
- Large complex composed of two distinct components
F1 and F0 - The F1 complex is a peripheral membrane protein
- The F0 complex is an integral membrane protein
- This protein can carry out two functions, the synthesis (ATP synthase) and breakdown (ATPase) of ATP
